Immobilization of planktonic algal spores by inkjet printing

The algal cell immobilization is a commonly used technique for treatment of waste water, production of useful metabolites and management of stock culture. However, control over the size of immobilized droplets, the population of microbes, and production rate in current techniques need to be improved. Here, we use drop-on-demand inkjet printing to immobilize spores of the alga Ecklonia cava within alginate microparticles for the first time. Microparticles with immobilized spores were generated by printing alginate-spore suspensions into a calcium chloride solution. We demonstrate that the inkjet technique can control the number of spores in an ejected droplet in the range of 0.23 to 1.87 by varying spore densities in bioink. After the printing-based spore encapsulation, we observe initial sprouting and continuous growth of thallus until 45 days of culture. Our study suggest that inkjet printing has a great potential to immobilize algae, and that the ability to control the number of encapsulated spores and their microenvironments can facilitate research into microscopic interactions of encapsulated spores.

(Ca 2+ ) or magnesium (Mg 2+ ) ions. Especially G blocks in alginate contributes to form strong and reversible crosslinking networks so called "egg-box structure" (Fig. 1) 33,34 . Owing to the unique egg-box structure, alginate has been used for various coating applications in cosmetics, food, biomedical/pharmaceutical purposes, and Li-ion batteries [34][35][36] . Since alginates are mainly isolated from microalgae including E. cava, alginate was selected as a polymer for immobilizing E. cava spores with the inkjet printing technique.
The objective of this work is to prove the capability of the inkjet printing technique with alginate for immobilization of algal spores of E. cava. We measured the viscosities of alginate-spore suspensions and assessed the long-term stability of droplet ejection and jetting characteristics to evaluate inkjet-printability of the suspensions. Then, we monitored the numbers of spores in droplets to quantify the controllability of delivery. Finally, the growth of the immobilized spores in PESI/GeO 2 medium was quantified to reveal a compatibility of the immobilization technique of algal spores.

Materials and Methods
Sodium alginate-spore suspension preparation. Sodium alginate was purchased from MP Biomedical LLC, USA. Reproductive thalli of E. cava were collected from the coast of Jeju Island, Korea, then shipped to the laboratory in an ice box. The thalli were washed with tap water to remove salt, sand and microorganisms. The middle part of thallus that includes sporangium was collected and the pieces were placed in sterilized seawater to liberate spores. The spore suspension was filtered using absorbent cotton. 2% (w/v) sodium alginate solution from brown algae (Sigma Aldrich, USA) was autoclaved for 20 min at 120 °C, cooled at room temperature and filtered using 0.45-µm syringe filter. The solution was mixed with the spore suspension to prepare a final 0.5% (w/v) sodium alginate solution with concentrations of 0.125 × 10 6 , 0.500 × 10 6 or 2.00 × 10 6 cells ml −1 .
Viscosity measurement. Shear viscosities of the suspensions were measured using a cone-plate rotational viscometer (DV2T, Brookfield AMETEK, US) at room temperature. The viscometer has a rotating cone with a radius of 2.4 cm and a stationary plate. Sodium alginate-spore suspension (0.5 ml) was placed between the plate and the cone. Shear rates between 50 s −1 and 1,500 s −1 were applied for 60 s; the viscosities were derived by averaging data of the final 10 s (n = 3).
inkjet printing of sodium alginate-spore suspensions. A DOD inkjet printing system (Jetlab II, MicroFab Technologies, Inc., USA) was used to print the sodium alginate-spore suspensions. A piezoelectric inkjet nozzle with a diameter of 80 µm was used and voltage pulse was applied at ±80 V in bipolar mode. The wave pulse had a rise time of 6.0 µs, dwell time of 13 µs, fall time of 9.0 µs, and echo time of 22 µs. Images of jet formation were acquired every 30 µs by a stroboscopic CCD camera equipped in the printing system, and a short-duration LED light.
Arrays of printed sodium alginate-spore suspensions were generated on glass slides. The drop spacing of the arrays was 400 µm and stage speed was 50 mm s −1 . Printed arrays were observed under an optical microscope and the average number of spores per drop was calculated (n = 30). fabrication and culture of spore-immobilized alginate microparticles. Sodium alginate-spore suspensions were printed using the inkjet printing system, the the printed drops were collected in a 35-mm Petri dish containing 1% (w/v) dissolved CaCl 2 in seawater. The seawater had been autoclaved for 20 min at 120 °C, cooled at room temperature and passed through a 0.45-µm filter. Printing was conducted at an ejection frequency of 1,000 Hz for 30 min. The solution was strirred using a 1-cm long magnetic stirring bar to prevent aggregation of the hydrogel particles. After printing, 70 µl of generated microparticle suspension was mixed with 200 µl of PESI medium 37

Result and Discussion
Strategy for fabrication of spore-immobilized alginate microparticles. Figure 2 illustrates the piezoelectric drop-on-demand inkjet printing system to generate alginate microparticles containing spores. When a voltage pulse is applied to a piezoelectric actuator of an inkjet nozzle, the nozzle ejects a series of picoliter droplets of sodium alginate-spore suspensions, which sink into a CaCl 2 solution in a receptacle. As soon as a droplet impacts on the surface of the solution, Ca 2+ enters guluronate blocks of alginate molecule, thus forming junctions with adjacent guluronate blocks 38 . As a result, alginate-based droplets are crosslinked and transformed into hydrogel microparticles that encapsulate spores. The solution in the receptacle was magnetically stirred to prevent aggregation of hydrogel particles (Supplementary Fig. 1).
Viscosities and jet formations of sodium alginate-spore suspensions. Viscosities of sodium alginate-spore suspensions was measured in the range of shear rate between 5 × 10 1 and 10 3 s −1 to examine their printability. All of the suspensions had viscosity <20 mPa•s, which is suitably low for stable printing (Fig. 3a). The spore density did not significantly affect viscosities due to the very small size of spore (several microns) and the low range of spore densities (0.125 × 10 6 ml −1 to 2.00 × 10 6 ml −1 ). Then, we observed their jetting behaviors for 2 hours. No clogging of the nozzle was observed during the experiments. Their representative high-speed images of jet formation are shown in Fig. 3b. With the same driving waveform and the negligible difference in shear viscosity, drop velocity was observed to decrease as spore density increased. We speculate that the naturally secreted component by E. cava spores inside the suspensions might slightly increase the elasticity of bioink.

Average number of spores per drop.
In order to quantify the controllability of delivery of spores, we investigated the number of spores in a droplet by printing dot arrays of sodium alginate-spore suspensions onto the glass substrate without crosslinking. Then, the number of spores in a drop was counted using microscope (Fig. 4a,b). Examination of the dot array showed increase in the average number of spores per drop as spore densities increased: 0.23 at 0.125 × 10 6 ml −1 , 0.67 at 0.500 × 10 6 ml −1 , and 1.87 at 2.00 × 10 6 ml −1 (Fig. 4f), and decrease in the size of dots as spore density increased (Fig. 4c-e). However, the average number of spores per drop did not increase as much as the spore density of the suspension did. The spore population may have increased the viscosity and elasticity of the suspension and reduced ejection volume of ink from the nozzle, as was also observed in a previous study 39 . This ability to control the number of encapsulated spores and their microenvironments demonstrates that the piezoelectric inkjet printing technique has possible uses as a method to study microscopic interactions of encapsulated spores.
Generation and culture of E. cava spores immobilized in alginate microparticles. Microparticles were generated using inkjet printing system with a receptacle containing CaCl 2 crosslinking solution as shown in Fig. 2. In order to monitor the morphology of spore-laden microparticles, they were observed using a microscope. The spores of E. cava were immobilized inside alginate microparticles (Fig. 5, white arrows). The crosslinking of alginate with calcium ion was proceeded quickly from the boundary of the printed droplet, the loss of spores is    www.nature.com/scientificreports www.nature.com/scientificreports/ Sprouting and growth of the entrapped spores were monitored for 45 d to prove a compatibility of the immobilization technique of algal spores. Spores immobilized in microparticles remained dormant until day 8 (Fig. 6), then a thallus began to grow. Average thallus length reached ~10 µm on day 14, and ~30 µm on day 21. After 45 days of culture, E. cava showed a bush-like morphology. The growing E. cava showed the gametophyte phenotype because the spores had been collected from sporophytes 10 . The results show that inkjet printing has applications for immobilization of algal cells. Furthermore, the growth trend was not significantly affected by spore density. This result indicates that E. cava spores at low to moderate density of within hydrogel microparticles could not significantly influence each other's thallus morphology. The range of the spore density at which such influence occurs should be determined in future studies.

conclusion
In this study, piezoelectric inkjet printing technology was used to immobilize spores of E. cava in sodium alginate suspensions. Viscosities and drop-formation behaviors demonstrated that they were suitable inks for the inkjet printing. The spatial density of spores may affect the viscosity and the elasticity of sodium alginate-spore suspension, and thereby affect jetting formation and lower-than-expected average number of spores per droplet. The printing technique allowed fabrication of spore-immobilized alginate microparticles. Culture of the microparticles yielded healthy gametophytes of E. cava without significant dependence on the spore density. These results show that inkjet printing has applications for immobilization of algal cells and single cell encapsulations.