J Appl Phycol DOI 10.1007/s10811-014-0411-6
Evaluation of sodium tripolyphosphate-alginate coating and re-calcifying on the entrapment of microalgae in alginate beads Ana B. Castro-Ceseña & M. del Pilar Sánchez-Saavedra
Received: 21 July 2014 / Revised and accepted: 11 September 2014 # Springer Science+Business Media Dordrecht 2014
Abstract Coating with polymers and re-calcifying have been done to retard the liberation of immobilized cells. Considering that sodium tripolyphosphate (TPP) is highly effective for chelating calcium ions, it is possible to postulate that if a layer of TPP is deposited over calcium alginate beads, the strong interaction between TPP ions and calcium may help in improving their cell holding capacity. In this study, we evaluate and compare the efficiency of cell entrapment between beads of alginate-immobilized microalgae Chlorella vulgaris, Acutodesmus (Scenedesmus) obliquus, and Synechococcus elongatus that were re-calcified, with those that were coated with varying ratios of alginate:TPP. Results indicate that recalcifying and coating do not limit the growth of the species studied. For C. vulgaris and A. obliquus, coating is more efficient for cell entrapment than re-calcifying. In contrast, for S. elongatus, re-calcifying is more effective than coating, suggesting a relationship between the cell size and the efficiency of cell entrapment. Keywords Microalgae immobilization . Sodium alginate . Sodium tripolyphosphate . Re-calcification . Entrapment
Introduction Immobilization of microalgae has demonstrated several advantages for wastewater treatment over the use of free-living microalgae. Immobilized microalgae result in higher reaction rates since cell density is greater; there is a reduced loosing of A. B. Castro-Ceseña : M. del Pilar Sánchez-Saavedra (*) Centro de Investigación Científica y de Educación Superior de Ensenada (CICESE), Ensenada, Baja California, Carretera Ensenada-Tijuana No. 3918, Zona Playitas, 22860 Ensenada, Baja California, Mexico e-mail:
[email protected]
cells because of their confinement, and the operational stability is better (Brouers et al. 1989; Mallick 2002). Carrageenan (Lau et al. 1997) and chitosan (Fierro et al. 2008) are natural polymers that have been widely used for microalgae immobilization. However, alginate is the polymer most commonly used in encapsulation systems (Vilchez et al. 2001; Cabrita et al. 2013). A major advantage of using alginate is that microalgae are not greatly affected, physically or chemically while being immobilized. Alginate is biocompatible and forms porous and transparent gels (Moreno-Garrido 2007). However, leaching is known to occur in alginate beads (Cheetam et al. 1979; Mohapatra and Hsu 2000; Serp et al. 2000; Ramachandran et al. 2009). For example, leaching in alginate-immobilized Dunalliela tertiolecta (size 10–12 μm) starts after 8 days of immobilization (Jiménez-Estrada 2011). In order to overcome the liberation of cells at early days of immobilization, coatings of polymer such as starch and polyethylene glycol (Jerobin et al. 2012), chitosan (Krasaekoopt et al. 2004), or alginate (Annan et al. 2008) have been used. A different approach used to diminish leaching has been recalcifying, which consists on hardening the beads again in fresh CaCl2 solution (Ruíz-Marín et al. 2010; Jiménez-Estrada 2011; Flores-Páez 2012; Ruíz-Güereca 2014). On the other hand, sodium tripolyphosphate (TPP) has been shown to be highly effective for chelating calcium ions (Ca2+) (Tarté 2009). Flores-Páez (2012) used for the first time solutions of different concentrations of TPP to immobilized cells of the microalga Scendesmus sp., taking advantage of the interaction of TPP with Ca2+ and applied a coat of TPP solution over Scenedesmus sp. immobilized in alginate beads. The author found that the higher the TPP concentration (1 %), the greater the reduction of the bead size and, when compared with uncoated beads and those chitosan-coated, the ones coated with TPP showed the smaller percent of cell liberation (4.2 %). Considering these previous findings, if a layer of TPP blended with alginate is deposited over a calcium alginate
J Appl Phycol
bead, it is plausible that the strong interaction between TPP ions and Ca2+ (from the calcium alginate matrix), together with the layer of alginate, may help in improving the cell holding capacity of the alginate beads. The purpose of inducing the interaction between Ca2+ and TPP is to retard the loss of Ca2+ from the beads, which is one of the factors that causes leaching of the immobilized cells. In this study, Chlorella vulgaris, Acutodesmus obliquus, and Synechococcus elongatus were chosen based on their good potential for aquaculture and biotechnology. These three species have been used successfully for wastewater nutrient removal (Lau et al. 1997; Fierro et al. 2008; Aguilar-May and Sánchez-Saavedra 2009). The three species can be grown in fresh water, but S. elongatus can also grow in seawater (Fierro et al. 2008; Aguilar-May and Sánchez-Saavedra 2009; Pérez-Coutiño 2011). The goal of this work is to compare the efficiency of cell entrapment between beads of alginate-immobilized C. vulgaris, A. obliquus, and S. elongatus that were recalcified and those that were coated with a layer of different ratios of alginate:TPP and evaluate if the size of the immobilized cells influences the cell holding efficiency.
Materials and methods The strains used in this study were Chlorella vulgaris (size 8 μm), Acutodesmus (Scenedesmus) obliquus (size 8 μm forming coenobia of 2 to 6 cells), and Synechococcus elongatus (size 1.8 μm and forming chains from 2 to 10 cells) which were obtained from the Microalgae Culture Collection from CICESE. The strains were maintained in nonaxenic cultures in 300-mL flasks in “f” medium (Guillard and Ryther 1962), using tap water for growth medium preparation. Cultures were kept at 20 °C with light intensity of 100 μmol photons m−2 s−1 under continuous light.
Formulations applied to beads after immobilization Alginate-immobilized C. vulgaris, A. obliquus, and S. elongatus were, individually for each species, coated with a layer of TPP-alginate, at different alginate:TPP ratios and stabilization times (Table 1). At the same time, a set of beads of each species was re-calcified with 2 % CaCl2 for 60 min, which are the optimum conditions previously found in our research group (Jiménez-Estrada 2011; FloresPáez 2012; Ruíz-Güereca 2014). Control beads (no coating or re-calcifying) for each species were kept throughout the experiments. Each set of beads corresponding to every formulation used was kept at the same light, culture medium, and temperature conditions described above.
Evaluation of the entrapment efficiency Chlorophyll a concentration was used to measure microalgae biomass and determined according to Parsons et al. (1984), with a HACH spectrophotometer (Model 4000 UV). The rate of production of chlorophyll a was then calculated in order to compare differences between the control beads produced with varying degrees of alginate:TPP coating and the re-calcified beads. The concentration of chlorophyll a during the assay, for every formulation used, is given by: ½Chl at ¼
Xt t¼0
½Chl at
ð1Þ
where [Chl a]t* is the sum of the chlorophyll a content reported at any time t* during the experiments. Plotting the cumulative Table 1 Formulations applied to beads of Chlorella vulgaris, Acutodesmus obliquus, and Synechococcus elongatus, at the eight day after immobilization in alginate
Immobilization procedure Entrapment of the algae in alginate-beads was performed as follows: 50 mL of non-axenic culture of each species were centrifuged at 4700 rpm for 5, 15, and 30 min for C. vulgaris, A. obliquus, and S. elongatus, respectively. Then, for each species, 0.1 mL of pellet was taken and then mixed with 10 mL of previously sterilized sodium alginate solution at 4 %. The alginate-immobilized microalgae beads were obtained by dropping the mixture into 2 % CaCl2 at room temperature using a burette. The beads were allowed to harden in the CaCl2 solution for 30 min (Chen 2001; Flores-Páez 2012). The beads were then washed with distilled water until pH was 8.0. The immobilized microalgae were kept for 6 days in 300 mL "f" medium at 20 °C with 100 μmol photons m−2 s−1 constant light intensity, before re-calcifying or coating on the seventh day.
Formulation
% alginate:% Stabilization Time of contact of beads TPPa coating time (min) with solution (min)
Control Re-calcificationb Alginate:TPP coating
– – 0.5:0.5 0.5:1.0 0.5:1.5 0.5:0.5 0.5:1.0 0.5:1.5 0.5:0.5 0.5:1.0 0.5:1.5
a
TPP=sodium tripolyphosphate
b
CaCl2, 2 % solution
– – 60 60 60 90 90 90 120 120 120
– 60 1 1 1 1 1 1 1 1 1
J Appl Phycol
chlorophyll a provides information about the amount of chlorophyll a produced at a certain time and total content of chlorophyll a produced over the time of the experiments. The rate of the increment of chlorophyll a content was expressed as the slope (b) of the exponential section of the plot of ln[Chl a]t* as a function of time. For all cases, data of cumulative chlorophyll a content fitted well (r2 >0.93) to an exponential curve. Measuring the chlorophyll a content, and its rate of production, may be useful for evaluating if the matrix of encapsulation provides adequate conditions for growing, but not necessarily for evaluating the entrapment efficiency. Therefore, the rate of liberation of cells from the beads was measured. The number of the liberated cells to the medium was counted using a hemocytometer (Neubauer, 0.1-mm deep). The rate of cell liberation was determined from the slope of the plot of the cumulative number of liberated cells as a function of time. Bead size Randomly selected beads were taken out from every formulation (Table 1) and the excess of water removed. The bead size was measured using a stereoscopic microscope (Olympus SZX7) and the ImagePro Discovery software. Statistical analysis The effect of re-calcifying and coating on bead size, for each of the encapsulated species, was analyzed by one-way analysis of variance (ANOVA). Statistical significance between treatments was determined by Tukey’s a posteriori test. Significance was determined at P0.95) an exponential tendency for the three species evaluated. The slope of the curve on the exponential section may be considered as the rate of chlorophyll a production. Therefore, A. obliquus showed the highest rate of chlorophyll a increase (bc =0.77), followed by C. vulgaris (bc =0.60) and S. elongatus (bc =0.45) (Fig. 1). Beads of alginate-immobilized C. vulgaris had, in general, a greater size (3.00 mm) than A. obliquus and S. elongatus (2.75 mm, for both species) (Fig. 1). Evaluation of the entrapment efficiency The three species reached, after re-calcifying or coating with a layer of alginate:TPP, a cumulative chlorophyll a content and a rate of production of chlorophyll a comparable to the control (Figs. 2, 3, and 4). The rate of liberation of cells
Comparison of the effect of re-calcifying and coating on bead size With regard to bead size, there was an apparent increase in the diameter of the beads after coating with a layer of alginate:TPP. For C. vulgaris and A. obliquus, the bead size for all the alginate:TPP formulations was significantly greater (Pc; mean values±SD, n=8)
a
TPP=sodium tripolyphosphate
b
CaCl2, 2 % solution
content curve. bF =slope of the liberated cells curve. r2c, r2F =coefficient of determination for chlorophyll a content and liberated cells, respectively. R=ratio between the rate of chlorophyll a increment and the rate of cell liberation, bc/bf
Bead size (mm) Formulation
% alginate:% TPPa, stabilization time
Chlorella vulgaris
Acutodesmus obliquus
Synechococcus elongatus
Control Re-calcificationb Alginate:TPP coating
– – 0.5:0.5, 60 0.5:1.0, 60 0.5:1.5, 60 0.5:0.5, 90 0.5:1.0, 90
2.96±0.07b 2.94±0.03b 3.07±0.10a 3.15±0.07a 3.12±0.06a 3.11±0.07a 3.10±0.06a
2.63±0.08b 2.61±0.04b 2.80±0.06a 2.78±0.07a 2.83±0.10a 2.87±0.08a 2.77±0.08a
2.69±0.13b,c 2.62±0.03c 2.87±0.10a 2.85±0.12a,b 2.86±0.12a,b 2.85±0.08a,b 2.80±0.07a,b
3.10±0.05a 3.10±0.06a 3.15±0.05a 3.10±0.05a
2.78±0.11a 2.87±0.07a 2.84±0.08a 2.78±0.08a
2.88±0.11a 2.97±0.15a 3.02±0.12a 3.04±0.14a
min min min min min
0.5:1.5, 90 min 0.5:0.5, 120 min 0.5:1.0, 120 min 0.5:1.5, 120 min
J Appl Phycol
With regard to the entrapment efficiency of each of the treatments applied, the rate of liberation of cells was similar for all the conditions used for alginate:TPP coating, and no tendency can be suggested as alginate:TPP ratio increases or stabilization time increases. However, for C. vulgaris, the 0.5:0.5, 60 min (alginate:TPP, stabilization time), and 0.5:1, 90-min formulation for A. obliquus, are the best conditions for holding the cells inside the beads. In the case of S. elongatus, the decline in the rate of liberation of cells from the recalcified beads is more apparent than for any other assay condition or even species. The size of the C. vulgaris and A. obliquus cells is about 8 μm, and the size of S. elongatus cells is approximately 1.8 μm. Considering that the formulations used in this study were the same, regardless of the cell size of the species, these results may be attributed to a relationship between the cell size of the entrapped microalgae and the characteristics of the polymeric matrix used for the entrapment, thus affecting the efficiency of cell holding. This can be better understood if we consider the differences between recalcifying and coating. While re-calcifying replaces the lost associations between the monomers of alginate, applying a coat of polymer provides a new barrier to hold the cells inside the beads, but does not restore the size of the existing pores of the capsules. In addition, it has been reported that stronger gels will tend to push more water out of the bead (Tarté 2009). Therefore, keeping in mind that syneresis or purge occurs on stronger gels, the beads that were re-calcified in this case, we may relate the conditions for coating with the efficiency of cell entrapment. This may help to explain why coating was more effective than re-calcifying for holding C. vulgaris and A. obliquus, the bigger size cells. If the polymeric matrix holding the cells is highly strong and syneresis takes place, leaching of the cells will increase. Therefore, a less strong gel, or coat, will reduce the associated syneresis (Tarté 2009). However, re-calcifying was more effective for holding S. elongatus, which suggests that the syneresis effect can be more apparent depending on the size of the encapsulated cells. Therefore, choosing the adequate treatment for the beads — i.e., applying a layer of polymer or re-calcifying—consideration of the cell size of the microalgae that will be encapsulated might make the difference between an efficient cell entrapment or not. With respect to the bead size, for C. vulgaris and A. obliquus—the species of bigger size (8 μm)—significant differences were found in the diameter of the beads, showing a greater increase after coating, which indicates that a layer of polymer has been deposited over the bead. In the case of recalcifying, no significant differences were found in bead size compared to the control. This is reasonable since re-calcifying does not imply any increment of the bead size, as obtained in this study. In conclusion, re-calcifying and coating with alginate:TPP do not cause any limitation to growth of the three species
studied. Coating with alginate:TPP, the bigger size microalgae (C. vulgaris and A. obliquus), is more efficient for cell entrapment than re-calcifying. In contrast, re-calcifying is more effective than alginate:TPP for the smaller alga (S. elongatus). These results suggest a relationship between cell size, characteristics of the polymeric matrix for immobilization, and entrapment efficiency. Selecting the proper conditions for entrapment considering the cell size might make the difference between an efficient entrapment or not. Acknowledgments This work was funded by the Consejo Nacional de Ciencia y Tecnología, CONACyT (Grant No. 130074) and by Centro de Investigación Científica y de Educación Superior de Ensenada (CICESE, project no. 623108). A.B. Castro-Ceseña received a postdoctoral fellowship from CONACyT (Grant No. 130074).
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