J Appl Phycol DOI 10.1007/s10811-015-0552-2
Effect of glycerol and PEGMA coating on the efficiency of cell holding in alginate immobilized Synechococcus elongatus Ana B. Castro-Ceseña & M. del Pilar Sánchez-Saavedra
Received: 31 January 2015 / Revised: 21 February 2015 / Accepted: 22 February 2015 # Springer Science+Business Media Dordrecht 2015
Abstract Synechococcus elongatus cells were immobilized in alginate beads, and the effects of increasing the crosslinker concentration from 2 to 4 % CaCl2 were evaluated, as well as the effects of coating the beads with either glycerol or poly(ethylene glycol) methyl ether methacrylate (PEGMA)— not previously reported for immobilized microalgae—to improve the holding time of the immobilized cells. S. elongatus cells remain metabolically active after coating with glycerol or PEGMA. There is an inverse relation between the glycerol concentration and the chlorophyll a content for the alginate beads cross-linked with 2 % CaCl2. PEGMA diminishes the rate of liberation of cells as its concentration increases, although results suggest the ability of S. elongatus to degrade PEGMA, which increases the growth rate of the liberated cells, because of PEGMA being used as carbon source. Keywords Synechococcus elongatus . Cyanobacterium . Immobilization . Coating . PEGMA . Glycerol
Introduction Alginate is a natural polymer commonly used for microalgae immobilization (Vilchez et al. 2001; Cabrita et al. 2013). However, increasing the holding time of immobilized cells still remains a challenge (Cheetam et al. 1979; Mohapatra and Hsu 2000; Serp et al. 2000; Ramachandran et al. 2009). An option to overcome the liberation of cells at early days of immobilization may be coating the beads with polymers. Several reports A. B. Castro-Ceseña : M. del Pilar Sánchez-Saavedra (*) Centro de Investigación Científica y de Educación Superior de Ensenada (CICESE), Carretera Ensenada-Tijuana No. 3918, Zona Playitas, 22860 Ensenada, Baja California, México e-mail:
[email protected]
on alginate have been focused on the effect on the mechanical properties and water vapor permeability of films cross-linked at several degrees of CaCl2 solutions (Blandino et al. 1999; Rhim 2004; Altenhofen da Silva et al. 2009) or cross-linked with chitosan glutamate (Remuñán-López and Bodmeier 1997). Other studies have been centered on analyzing the physicochemical and/or mechanical properties of alginate films plasticized with glycerol (Pavlath et al. 1999; Avella et al. 2007; Jost et al. 2014), and others have reported on the controlled release of antiseptic compounds (Liakos et al. 2013), the survival of probiotic bacteria after encapsulation in alginate coated gelatin beads (Annan et al. 2008), or the controlled release of biological compounds from alginate beads coated with starch or poly(ethylene) glycol (Jerobin et al. 2012). However, to the best of our knowledge, this is the first study that reports on glycerol and poly(ethylene) glycol coatings of alginate immobilized microalgae live cells. Synechococcus elongatus is characterized as a cosmopolitan cyanobacterium with a good potential for aquaculture and biotechnology (Matsunaga et al. 1988; Sasikala and Ramana 1994; Kämäräinen et al. 2012; Xu et al. 2013). In addition, given the small cell size of S. elongatus (1.8 μm), increasing the retention time of these cells represents a challenge. Therefore, the goal of this work is to immobilize S. elongatus cells in alginate beads, and evaluate the effects of increasing the cross-linker concentration from 2 to 4 % CaCl2, as well as the effects of coating the beads with either glycerol or poly(ethylene glycol) methyl ether methacrylate (PEGMA) to improve the holding time of the immobilized cells.
Materials and methods The cyanobacterium S. elongatus was obtained from the BMicroalgae Culture Collection from CICESE.^ S. elongatus was kept in non-axenic cultures in 300-mL flasks in Bf^
J Appl Phycol
medium (Guillard and Ryther 1962), prepared with sterilized tap water, as previously reported (Aguilar-May et al. 2007; Aguilar-May and Sánchez-Saavedra 2009; Castro-Ceseña and Sánchez-Saavedra 2014). The culture was maintained at 20 °C with 24 h constant light, at an intensity of 100 μmol photons m−2 s−1 as described by Aguilar-May (2006).
Immobilization and coating procedures S. elongatus was immobilized as follows: 50 mL of nonaxenic culture was centrifuged at 20 °C, 4000 rpm for 20 min. The supernatant was removed, and 0.1 mL of pellet was mixed with 10 mL of 4 % sodium alginate. Beads of alginate immobilized S. elongatus were obtained by dropping the mixture, using a burette, into a solution containing the coating agent and either 2 or 4 % CaCl2 (Table 1), at room temperature. Two different types of coating were evaluated: glycerol (99.5 %) ACS Reagent and poly(ethylene glycol) methyl ether methacrylate (PEGMA) (Mn 950), both from Sigma-Aldrich, Mexico. The beads of alginate immobilized S. elongatus were submerged into a glycerol solution containing CaCl2 (Table 1) for 30 min. Another set of beads was coated with PEGMA (containing CaCl2); in this case, the beads were immersed in the solution for 20 min (Table 1). The final volume of the solution containing the coating agent and CaCl2 was 6 mL Table 1 Conditions used for coating alginate immobilized Synechococcus elongatus Alginate (%)
Free culture Immobilization conditions
CaCl2a (%)
Coating material Glycerol (%)
PEGMA (mg mL−1)
Statistical analysis An analysis of variance of the main effects was performed in order to statistically evaluate the rate of increment of chlorophyll a (kc), the rate of growth rate of the liberated cells (kG), and the sizes of the beads between the different experimental conditions. Statistical significance between treatments was determined by Tukey’s a posteriori test. Significance was determined at P0.05) in bead size as the concentration of glycerol coating increases in the beads prepared with 4 % alginate and cross-linked with 2 % CaCl2
J Appl Phycol
(3.35±0.06, 3.40±0.07, 3.35±0.14 mm for 0.5, 1, 2 % glycerol, respectively) (Table 2). However, bead size diminishes significantly (P0.05) change as the concentration of PEGMA increases, neither when alginate is cross-linked with 2 % CaCl2 nor when 4 % CaCl2 is used (Table 2). Bead size significantly increases (P0.05) are found between the size of the beads that were cross-linked using 2 or 4 % CaCl2 after being coated. For example, the size of the beads cross-linked with 2 % CaCl2 and coated with 0.5 % glycerol (3.35±0.06 mm) is not significantly different (P>0.05) to the size of the beads cross-linked using 4 % CaCl2 and coated with the same glycerol concentration (3.43±0.08 mm). The same occurs to the beads coated with PEGMA. The beads cross-linked with 2 % CaCl2 and coated with 10 mg mL−1 PEGMA (3.04± 0.05 mm) are not significantly different (P>0.05) to those cross-linked with 4 % CaCl2 and coated with the same concentration of PEGMA (2.98±0.05 mm) (Table 2). Biomass growth There is a significant difference (P c > d). Mean values±SD, n=15 PEGMA poly(ethylene glycol) methyl ether methacrylate a
With respect to alginate
Bead
Coating
Alginate (%)
CaCl2a (%)
Glycerol (%)
4 4 4 4
2 2 2 2
– 0.5 1 2
4 4 4 4
4 4 4 4
– 0.5 1 2
Regarding the different coating conditions applied to the immobilized cells, chlorophyll a content tends to increase over time (Fig. 1). Comparing the response to a specific treatment condition, we found that when S. elongatus is immobilized in 4 % alginate and cross-linked with 2 % CaCl2, the chlorophyll a content diminishes as the concentration of the coating increases from 0.5 to 2 % glycerol (Fig. 1b). For the same conditions, kC significantly diminishes (P e > f). Mean values ± SD, n=3. N.C. not calculated since chlorophyll a content (biomass) was detected until the 30th day of the experiment. No cells were found in the medium due to the low biomass growth of the immobilized cells. N.E. not evaluated due contamination with other microalgae detected in the medium; therefore, chlorophyll a was not measured, PEGMA poly(ethylene glycol) methyl ether methacrylate a
With respect to alginate
b
Rate constant for the increment of chlorophyll a content in beads
c
Growth rate constant of the liberated cells to media
d
Mean punctual data corresponding to the 30th day
studies have shown that the matrix of alginate beads crosslinked with CaCl2 for neem oil encapsulation became more compact and rigid after coated (starch or poly(ethylene)glycol, PEG) (Jerobin et al. 2012). This compaction might contribute to counterbalance the effect on the bead size after increasing the cross-linker concentration in the coated beads, suggesting an interaction between the calcium-alginate from the beads and the coating materials. Measurements of chlorophyll a content can provide information about biomass growth, indicating the suitability of each of the treatments applied and how the immobilization and coating conditions affect the increment of S. elongatus biomass. The increase in chlorophyll a content in the immobilized cells is apparent with respect to free culture. This is an expected finding that has been previously reported (Lau et al. 1997; Aguilar-May and Sánchez-Saavedra 2009) and may be attributed to an increase of cell biomass inside the beads, as well as a compensating mechanism of the selfshading and gel matrix screening effects (Lau et al. 1997). This result indicates that cells keep being metabolically active as showed by the continued synthesis of chlorophyll (Lau
et al. 1997), even for those treatments where the higher polymer concentration, either glycerol or PEGMA, was used. The results obtained show an inverse relation between the glycerol concentration and the chlorophyll a content for the alginate beads cross-linked with 2 or 4 % CaCl2, and these results are corroborated by the kC calculated for chlorophyll a content. Therefore, as the concentration of the glycerol coating increases, the smaller chlorophyll a content reached, and the slowest the production of chlorophyll (low kC). Chlorophyll content is commonly used as a physiological indicator of the photosynthetic and metabolic activity of microalgae (Lau et al. 1997); keeping this in mind, the reduction on the rate of production of chlorophyll may be interpreted as a lowering of the metabolism of the cells as the concentration of glycerol increases. All the beads of any of the treatments were transparent; therefore, light was available to the immobilized cells. Thus, the lowering of the metabolism of the immobilized S. elongatus may be attributed to a decreased permeability of the beads to the nutrients and oxygen from the surrounding f medium, as the glycerol concentration increases.
J Appl Phycol Fig. 2 Number of cells (log2 cells mL−1) liberated from the 4 % alginate immobilized Synechococcus elongatus to the media, using a 2 % CaCl2 or c 4 % CaCl2 as cross-linker, and glycerol for coating, b 2 % CaCl2 as cross-linker, and PEGMA for coating. a, c 0 % (black square), 0.5 % (red circle), 1 % (pink up triangle), and 2 % (blue down triangle) glycerol. b 0 mg mL−1 (black square), 5 mg mL−1 (red circle), 10 mg mL−1 (pink up triangle), and 20 mg mL−1 (blue down triangle) PEGMA
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Days Fig. 3 Plots of the pH of a free culture medium of Synechococcus elongatus. Medium with beads of S. elongatus immobilized in 4 % alginate, cross-linked with b 2 % CaCl2 or d 4 % CaCl2 coated with glycerol. Medium with beads of S. elongatus immobilized in 4 % alginate, cross-linked with c 2 % CaCl2 or 4 % CaCl2 coated with
12 16 20 24 28 32
Days PEGMA. b, d 0 % (black square), 0.5 % (red circle), 1 % (pink up triangle), and 2 % (blue down triangle) glycerol. c, e 0 mg mL−1 (black square), 5 mg mL−1 (red circle), 10 mg mL−1 (pink up triangle), and 20 mg mL−1 (blue down triangle) PEGMA
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No biomass, i.e., chlorophyll a, was detected in the uncoated beads made of 4 % alginate and cross-linked with 4 % CaCl2, until the end of the study (30th day). Growth of cells was detected in beads coated with 0.5, 1 % glycerol, and 5, 10 mg mL−1 PEGMA. It is known that glycerol, as a plasticizer, reduces the number of physical cross-links between the macromolecular chains of the polymer gel leading to a disaggregating effect on the matrix structure, introducing flexibility (Coffin and Fishman 1994; Gupta et al. 2014). An increase in plasticizer concentration will increase the water absorbed by the gel (Zactiti and Kieckbusch 2006; Jost et al. 2014). Therefore, our results suggest an interaction between the calcium-alginate beads and the coating solution, producing a plasticizer effect in the coated beads. As a result, the nutrients from the medium could be more available to the coated beads leading to growth of biomass. A lower chlorophyll a content was measured in the beads cross-linked with 4 % CaCl2 compared to those with 2 % CaCl2. This may be a result of the highly cross-linked alginate matrix gel when 4 % CaCl2 was used. Previously, the permeability to water was reported to reduce in alginate films—used as antimicrobial edible coatings on food—as an effect of the increased cross-linking degree with calcium ions (Benavides et al. 2012); therefore, the smaller values of chlorophyll, i.e., biomass, in the 4 % CaCl2 cross-linked beads may be attributed to a reduced permeability to nutrients, which may be due to a combined effect between (1) the highly cross-linked bead and (2) the more compacted and increased rigidity of the beads due to the high concentration of the coating material (Jerobin et al. 2012) that reduced the rate of diffusion of nutrients to the interior of the beads, leading to a slower biomass growth, as a consequence. However, the characteristics of the polymeric matrix used for immobilization might produce different results in terms of entrapment efficiency, depending on the size of the immobilized microalgae (Castro-Ceseña and Sánchez-Saavedra 2014), and their exposure to chelating agents or antigelling cations, present in media such as estuaries (Moreira et al. 2006), depending on the application of the immobilized cells. The saline water of estuaries leads to gel instability in alginate immobilized microalgae. In such cases, increasing CaCl2 from 2 to 4 % CaCl2 would compensate the instability caused by the exposure to saline water (Moreira et al. 2006). Therefore, characteristics such as salinity or presence of chelating agents in the medium might influence the effect of increasing the concentration of CaCl2 as cross-linker or hardener. A comparable interpretation can be given to the results obtained for the beads coated with PEGMA, excepting the beads of alginate cross-linked with 4 % CaCl2. In this case, the high rigidity of the beads together with the increased compactness (Jerobin et al. 2012), because of the PEGMA coating, diminishes the availability of nutrients to the immobilized cells leading to a deficiency of biomass growth.
Given the low chlorophyll content measured in the 2 % CaCl2 cross-linked alginate beads, we expected a small number of free cells in the f medium. However, the opposite occurs. As previously mentioned, an interaction between the alginate gels and the coating solution occurs, even though the gels are cross-linked, leading to plasticization of the gels when immersed in the glycerol or PEGMA solutions for coating. This is plausible considering the degree of unsolubilization of the beads. The higher the cross-linker concentration, the higher the degree of unsolubilization of the alginate gels (Rhim 2004). This may be the reason why the low cross-linked beads (2 % CaCal2) were more prone than those highly cross-linked (4 % CaCl2) to interact with the coating solution, allowing a certain degree of plasticization as a consequence. This plasticization of the alginate gel increases the permeability of the beads and allows the liberation of cells to the medium, which may explain the direct relation between glycerol concentration and the rate growth of the liberated cells (kG) obtained in this work, the higher the glycerol concentration, the higher plasticization of the beads and the more cells liberated. In addition, some of the glycerol coating solution might be accessible to the free cells and be used as carbon source by the microalgae (Kong et al. 2013), increasing the kG as a consequence. In the case of the PEGMA-coated beads, chlorophyll a content diminishes as the PEGMA concentration increases, and the liberated cells to the medium decrease also, which is the expected effect. However, the ratio kC/ kG is greater for the highest PEGMA coating concentration compared to that of the uncoated beads, suggesting that PEGMA may also be degraded and used as a carbon source by S. elongatus. But, reports on the assimilation of polyethylene glycols have mainly been focused on aerobic bacteria (Ogata et al. 1975; Watson and Jones 1977; Hosoya et al. 1978). This is the first work suggesting the ability of S. elongatus to degrade PEGMA. Although an organic substrate (glycerol or PEGMA) was used, the associated bacterial flora of S. elongatus was not observed in the culture media, under visual analysis with the microscope, throughout the study. Finally, microalgae, by photosynthesis, raise the pH of their medium, regardless of the carbon source (Verduin 1964). The biomass growing trend discussed above is supported by the pH tendency obtained here, which shows an increment of the pH of the medium throughout of the experiment, as occurred with the biomass trend. In conclusion, S. elongatus cells keep being metabolically active as showed by the continued synthesis of chlorophyll, even for those treatments where the higher polymer concentration, either glycerol or PEGMA, was used. And, no significant differences (P > 0.05) are found between the size of the beads that were cross-linked using 2 or 4 % CaCl2 after being coated.
J Appl Phycol
The results show an inverse relation between the glycerol concentration and the chlorophyll a content for the alginate beads cross-linked with 2 % CaCl2. Therefore, as the concentration of the glycerol coating increases, the smaller the chlorophyll a content reached, the slowest the production of chlorophyll. With regard to the beads cross-linked with 4 % CaCl2, no chlorophyll is detected, until the last days of the study, indicating the reduction of permeability to nutrients due to a highly cross-linked gel matrix. However, an interaction between the alginate gels and the coating solution occurs, even though the gels are cross-linked, leading to plasticization of the gels when immersed in the glycerol or PEGMA solutions for coating. Finally, when comparing glycerol and PEGMA as coating materials, under the conditions used here, the results indicate that PEGMA diminishes the rate of liberation of cells as its concentration increases, in spite of results suggest the ability of S. elongatus to degrade PEGMA, which increases the microalgae growth rate because of being used as carbon source. Acknowledgments This work has been funded by the Consejo Nacional de Ciencia y Tecnología, CONACyT (Project SEPCONACyT 2009-01-130074) and by Centro de Investigación Científica y de Educación Superior de Ensenada, CICESE (project 623108). A.B.. Castro-Ceseña acknowledges her postdoctoral fellowship from CONACyT (Project SEP-CONACyT 2009-01-130074).
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