3D Printing of Functional Microalgal Silk Structures for Environmental

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3D printing of functional microalgal silk structures for environmental applications Siwei Zhao, Chengchen Guo, Allison Kumarasena, Fiorenzo G Omenetto, and David L Kaplan ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.9b00554 • Publication Date (Web): 25 Jul 2019 Downloaded from pubs.acs.org on July 29, 2019

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ACS Biomaterials Science & Engineering

3D printing of functional microalgal silk structures for environmental applications Siwei Zhao1,†, Chengchen Guo1, Allison Kumarasena1, Fiorenzo G. Omenetto1,2,3,4 and David L. Kaplan1,* Addresses: 1. Department of Biomedical Engineering, Tufts University, Medford, MA 02155, USA 2. Silklab, Department of Biomedical Engineering, Tufts University, 200 Boston Avenue, Suite 4875, Medford, MA 02155, USA 3. Department of Electrical and Computer Engineering, Tufts University, Medford, MA 02155, USA 4. Department of Physics, Tufts University, Medford, MA 02155, USA † Present address: Department of Surgery, University of Nebraska Medical Center, 985965 Nebraska Medical Center, Omaha, NE 68198, USA Corresponding Author * David L. Kaplan, email: [email protected]

Abstract Silk protein-based hydrogel materials suitable for hosting living microalgae due to the biocompatibility and ambient conditions gelation were developed. The silk was selected due to its robust mechanical properties, safe and compatible utility, green sourcing, and versatile materials formation. Through a series of assessments the mechanics and gelation kinetics of the 1 ACS Paragon Plus Environment

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hydrogel materials were optimized for three-dimensional (3D) printing. Silk hydrogel structures containing a marine microalgal strain, Platymonas sp. were printed and these structures supported cell proliferation for at least four weeks and consistent photosynthetic activity for more than 90 days, the limits of the study timeframe. This long-term cell viability and function suggests that these systems may be suitable for a broad range of applications, such as oxygen replenishment and carbon dioxide reduction towards a green, healthier indoor environment.

Key words: Silk, Microalgae, 3D printing, Photosynthesis, Green technology

Introduction Microalgae are a large group of unicellular organisms that are capable of photosynthesis. Microalgae are well known for their heartiness or robustness and sustainability, as they grow rapidly and they adapt well to harsh culture conditions, such as wastewater (1, 2). Further, the culture conditions utilized for microalgae are environmentally friendly due to the low energy consumption and low waste production (2). These advantages have resulted in broad interest in microalgae for a broad range of applications. Microalgae are most commonly used in biomass production to converts sunlight and carbon dioxide to biofuels and hydrogen gas (3). This process is environmentally friendly as it does not involve any toxic chemicals. Microalgae can also produce metabolic substances that are valuable for industries such as in food production and pharmaceuticals. For example, microalgae are a source of protein, vitamins and minerals and thus are frequently consumed as nutritional supplements for both humans and animals. Substances with antibacterial, antifungal and antiviral activities have also been extracted from 2 ACS Paragon Plus Environment

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microalgae and used in medical applications (4). Microalgae have also been used in wastewater treatment. Here, compared to conventional methods such as chemical oxidation, microalgaebased wastewater treatment is more cost-effective due to lower energy consumption, reduced waste generation and algal biomass as a byproduct for later use as biofuels (2). Certain microalgae species exhibit high sensitivity to environmental changes, which has led to the development of microalgae-based biosensors for water quality and monitoring contamination (5, 6). Recently, large scale microalgae cultures have been established next to electric power plants to directly sequester CO2 generated by these plants, as a low-tech and low-cost carbon capture method (7). Although microalgae hold potential for many industrial, environmental and medical applications, the small size of single microalgal cells presents significant challenges to scale up for practical systems. Microalgae have to be immobilized in these systems to maintain population and function (5, 8, 9). Immobilization also allows ease of handling and collection of cellular products. In addition, proper immobilization minimizes liberation of microalgae into the surrounding environment, which can otherwise cause contamination to natural water systems particularly where the microalgae can outgrow native species (2). For certain applications, such as waste water treatment, immobilized microalgae have improved process efficiency compared to free cells (5). A variety of materials have been studied as host matrices for microalgae immobilization. Synthetic polymers, such as polyacrylamide, photo-crosslinkable polymers and polyurethanes, which exhibit long-term stability, have limited degradability, environmental compatibility and may present toxicity to living algal cells (5). Natural protein-based hydrogel materials (e.g., gelatin, collagen) are cell compatible, but have weak mechanical properties, and are costly (5). Natural polysaccharide hydrogels (e.g., agar/agarose, carrageenan, alginate), currently the most 3 ACS Paragon Plus Environment


commonly used substrates, exhibit significant advantages, including low cost and relative robust mechanics. Recent work by Krujatz and colleagues (8, 9) studied culture conditions (temperature and mode of illumination) for microalgae immobilized in 3D printed alginate hydrogel matrices This work achieved high algal viability and growth rate even under non-optimal conditions. Microalgal proliferation in alginate hydrogel matrices was observed over 12 days and photosynthesis was observed for 48 hours. A calcium ion-containing medium was used for the cultivation of the microalgae-encapsulating alginate hydrogels. 3D printing was used to fabricate multi-material systems that incorporated both microalgal cells and mammalian cells to demonstrate the feasibility of using microalgal hydrogel structures to supply oxygen to mammalian tissues cultured in close vicinity. However, alginate hydrogels are relatively unstable in aqueous environments (10, 11), while other polysaccharide-based hydrogels require high temperature for processing (e.g. agar/agarose). These disadvantages have limited the species of microalgae that can be immobilized within the gels, as well as the utility of these systems for longer-term applications as in the current work reported here. Silk is natural protein-based polymer with excellent biocompatibility, robust mechanical properties and available in industrial scales via the textile industry at reasonable costs (12-14), which makes it suitable for interfacing with biological systems, such as living cells and tissues, as well as for environmental applications. Our lab recently developed an enzymatically crosslinked hydrogel material based on silk (15, 16). The tyrosine residues on silk fibroin chains crosslink to form di-tyrosine bonds in the presence of horseradish peroxidase and hydrogen peroxide. The gelation process is conducted at ambient conditions in aqueous solution, along with cells, presenting no hazard to the biological materials or the environment. By adjusting the silk concentration, molecular weight and post crosslinking treatment, a range of mechanical 4 ACS Paragon Plus Environment

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stiffness spanning over three orders of magnitude (kPa to MPa) was achieved, which highlights the tunability of such material (16). We have demonstrated the 3D encapsulation of mammalian cells and their long-term culture in these hydrogels, which supports their excellent cytocompatibility (15-17). Importantly, these chemically crosslinked silk hydrogel materials showed long-term stability in physiological cell culture environments (16), providing unique benefits over commonly used alginate hydrogel materials. Therefore, silk hydrogel materials are particularly suitable for applications that require sustained device stability and functions. Finally, these materials can be enzymatically digested on demand (18). All of these features, from mechanical tunability, biocompatibility and long-term stability make silk hydrogel suitable for exploration for microalgae immobilization. The goal here was to study microalgae encapsulated silk hydrogel materials as possible matrices for long term function. The focus was on long-term stability, continuous cell growth and maintenance of photosynthetic activity. 3D printing was used to generate microalgal silk hydrogel structures with complex 3D geometries, while keeping the microalgae alive and functional. These microalgal silk hydrogel structures could be suitable for small scale environmental applications, such as CO2 reduction and oxygen replenishment in confined environments, such as in residential/commercial buildings and small fisheries.

Materials and Methods Materials A marine microalgae strain, Platymonas sp. was used for the study. Platymonas is a microalgal strain that has been frequently utilized in photosynthesis-related studies in both free and 5 ACS Paragon Plus Environment


immobilized formats (19-21). The microalgae and its medium were obtained from Carolina Biological Supply Company (Burlington, NC, USA). A commercial seawater microalgal medium was used for this study, which contained NaNO3, thiamine, biotin, vitamin B12, and other trace salts. Tris buffer was used to maintain its pH at 8. Raw silk cocoons produced by Bombyx mori silkworms were obtained from Tajima Shoji Co (Yokohama, Japan). Sodium carbonate, lithium bromide (LiBr), horseradish peroxidase (HRP), hydrogen peroxide (H2O2) and hydroxypropyl methylcellulose (HPMC, molecular weight ~26 kDa) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dialysis cassettes was purchased from Fisher Scientific (Pittsburgh, PA, USA). 3D printing syringes were purchased from Nordson Medical (Marlborough, MA, USA). Blunt needles were purchased from McMaster-Carr (Robbinsville, NJ, USA). Food coloring was obtained from a local grocery store. Silk processing Our previously established protocol was used to prepare the silk fibroin solution. Briefly, silk cocoons were diced into 1cm2 pieces. The cocoon pieces were then degummed by boiling in 0.2% w/w sodium carbonate solution for 30 min in order to remove the sericin. The degummed silk fibers were rinsed in copious amount of water and allowed to dry for at least 12 hours. 9.3M LiBr solution was then used to dissolve silk fiber into fibroin solution at 60oC for 4 hours. The silk fibroin solution was dialyzed against deionized (DI) water in dialysis cassettes with a 3,500 molecular weight cutoff. A 150 ml silk fibroin solution was dialyzed against 4 L DI water for three days with at least 6 water changes (at 1, 3 and 6-hours, the morning and the afternoon of the second day and the morning of the third day) to reduce the LiBr content. During the dialysis, the conductivity of the water was monitored using a handheld conductivity meter. The dialysis was considered complete when the conductivity of the water was less than 0.2 µS/cm (22). The 6 ACS Paragon Plus Environment

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conductivity of the silk solution at the completion of the dialysis was 325 ± 7 µS/cm. Finally, the fibroin solution was centrifuged at 9,000 rpm at 4oC for 20 min twice and filtered through a filter with 5µm pore size to remove undissolved residues. The resulting solution contained only silk fibroin at 6% w/v and water. The silk fibroin solution was concentrated in dialysis cassettes in a fume hood to reach a concentration of 26% w/v, which was used later to prepare microalgae/silk mixtures for the hydrogel 3D printing. Microalgae culture Microalgae were cultured according to the protocol recommended by the supplier. In brief, Platymonas sp. was cultured in suspension in sterile Erlenmeyer flasks at room temperature. Cool-white fluorescent lights at 2152 to 4304 lux were used for the initial 7 to 10 days to allow microalgae to grow. Light intensity was then lowered to 538 to 1076 lux to slow the growth for storage. A 16-hour light:8-hour dark cycle was used. Constant air bubbling with an air stone was used to supply oxygen to the medium to keep an average oxygen concentration of 11.3 mg/L. Microalgae concentration was calculated using a hemocytometer at the time of harvesting. 3D printing An Inkredible 3D bioprinter (Cellink, Boston, MA) was used to print microalgae/silk inks. The Inkredible bioprinter uses stable air pressure to extrude ink out of nozzle, allowing more constant ink flow and thus better consistency in printing quality. To prepare microalgae/silk inks (i.e. the precursor of microalgal silk hydrogels), microalgae were harvested and concentrated at 1,200 rpm for 5 minutes. The concentrated microalgae solution was then mixed with the 26% w/v silk fibroin solution at 2:3 volume ratio, so the final silk concentration was 15.6% w/v. For ink mixtures that did not contain microalgae, only microalgal medium was added at this step. The

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microalgae/silk mixtures were then mixed with 15% w/v hydroxypropyl methylcellulose (HPMC) solution at various weight ratios (3:7, 4:6, 5:5 and 6:4, which are referred to as silk-toHPMC ratio later) to increase the viscosity of the ink mixture to facilitate 3D printing. Finally, HRP was added to the ink mixture at different final concentrations (60, 120 and 180 unit/ml). The ink mixtures were then loaded in syringes that were specifically designed to work with the Inkredible bioprinters. The ink-loaded syringes were centrifuged at 2,000 rpm for 3 minutes to remove any air bubbles generated during the mixing of the ink. 3D models were designed with 3ds MAX (Autodesk, San Rafael, CA, USA). The models were sliced and translated to G-code using Repetier-Host (Hot-World GmbH & Co. KG, Germany). A 0.5 mm layer height and 0.5 mm nozzle diameter were used for the printing. Blunt needles were used as printing nozzles. The air pressure for 3D printing was adjusted based on the viscosity of ink mixture to maintain proper ink flow so the thickness of each layer equaled the layer height (i.e., 0.5 mm). The ink was extruded into a medium (DI water for ink without microalgae, or microalgal medium for ink with microalgae) containing 0.01% w/w H2O2 to initiate crosslinking immediately after printing. This concentration of H2O2 was selected to allow efficient crosslinking to form the gels, while maintaining high cell survival (15, 23). In order to prevent premature gelation in the nozzle, ink extrusion was never stopped during the printing process. When the nozzle was moved from one layer to another layer, or from one location to another location, the extrusion was switched to a much slower speed. After the microalgal silk hydrogel was sufficiently crosslinked, the H2O2 containing medium was replaced with microalgal medium to support cell proliferation. Characterization of 3D printed silk hydrogel materials Ink mixtures at different silk-to-HPMC ratios were evaluated by rheometry (TA instruments, ARES-LS2) to characterize the dynamic viscosity at room temperature. A cone and plate 8 ACS Paragon Plus Environment

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geometry (diameter = 25 mm, cone angle = 0.0994 rad, gap = 50 µm) was used for the viscosity measurements. Ink mixtures were challenged with a shear rate ramp from 0.001 s-1 to 1000 s-1, with 12 seconds of constant flow at each shear rate. The resulting shear stress was used to calculate the viscosity. The data points with a torque value of less than 0.02 g×cm were not analyzed because they were below the detection limit of the force transducer. HRP with different final concentrations (60, 120 and 180 units/ml) was added to these ink mixtures, which were 3D printed in 0.01% H2O2 solution into a disc shape with 1.35 cm diameter and 2 mm height. The hydrogel discs were fully crosslinked overnight in H2O2 solution before they were tested for Young’s modulus by Instron (model 3366, Norwood, MA, USA). Mechanical compression and 40% maximum strain were used in these evaluations. The loading rate was 0.2 mm/minute. Young’s modulus was calculated at 20% strain. To characterize gelation kinetics of printed inks, ink mixtures were 3D printed in black 96 well plate in H2O2 solution. Immediately after printing, the intrinsic fluorescence of crosslinked phenolic groups at 415 nm was measured using a 315 nm excitation wavelength until a plateau was reached. The fraction of the maximum intensity, after subtracting background with H2O2, is reported as the degree of gelation. To measure the optical transparency of the printed structure, ink mixtures with 180 unit/ml HRP were printed into a disc shape with 2 cm diameter and 0.5 mm height in H2O2 solution and allowed to crosslink overnight. The optical absorbance was then measured from 350 nm to 750 nm, at 50 nm intervals, using a plate reader (SpectraMax M2, Molecular Devices). For all hydrogel materials characterization mentioned above, microalgae were not added. Characterization of microalgae proliferation and photosynthesis in 3D printed hydrogel structures



All microalgal silk hydrogel structures were printed using an ink mixture with 6:4 silk to HPMC ratio and 180 unit/ml HRP concentration. This formula was selected due to optimal ink viscosity, gelation speed, mechanical stiffness and optical transparency. For microalgal proliferation evaluations, a 0.5 mm thick gel was printed in each well of a 24-well plate. The initial microalgal concentration was 7.5x106 cells per milliliter of hydrogel. This low initial concentration was selected to allow room for microalgal proliferation and to facilitate easier cell counting. The microalgal silk hydrogels were then imaged using phase-contrast bright-field microscopy at days 0, 2, 6, 10, 17 and 30, and all cells that were in focus were counted to quantitatively calculate algal proliferation rate. The proliferation rate is presented as average daily increase of cell number (%), calculated by averaging the percent increase of cell numbers since the prior observation over the number of days since the prior observation. The commonly used live (e.g. calcein) and dead (e.g. ethidium homodimer and SYTOX Green) cell stains were not used in this study because silk biomaterials have strong auto-fluorescence in both green and red channels (24-26). Because the silk/HPMC hydrogels studied in this work had a high silk content (up to 9.36% w/v), the auto-fluorescence of silk significantly interfered with cell staining signals. Biochemical oxygen demand (BOD) bottles were used to characterize the photosynthesis of microalgal silk structures. The BOD bottles have a special shoulder design, which helps to push out all air from the bottle when a sample solution is being added into the bottle. Microalgal hydrogel sheets 5 cm wide, 10 cm long and 2 mm thick were printed and transferred to a BOD bottle. The initial microalgal concentration in these sheets was 5x107 cells per milliliter of hydrogel. Then 30 ml microalgal medium was added in each bottle, which was sufficient to push out all of the air and to form a water seal at the cap of the BOD bottle to prevent air exchange. These steps ensured that all oxygen produced was in the medium. A 16-hour light:8-hour dark


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cycle was used for this part of the study. The oxygen concentration of the microalgal medium was measured once every 3 days using a needle-type optical oxygen sensor and a Microx 4 meter (both from PreSens, Germany) after 12 hours of light. After the measurement, the medium was replaced and the fresh medium was measured again. The oxygen concentration was reported as mg/L. The photosynthesis of free microalgae with the same cell number was used as a positive control, and microalgae-free silk/HPMC hydrogel was used as a negative control. Statistics Young’s modulus of hydrogels, microalgal proliferation rate and oxygen production of microalgal silk structures were tested in duplicates. For microalgal proliferation, three images per sample per time point were used for analysis. The gelation kinetics was tested in triplicates. These data are presented as the mean with standard deviation (SD). The dynamic viscosity and the optical transmittance were based on one independent measurement.

Results and Discussion Hydrogel 3D printing has recently attracted attention in both materials and biomedical research fields due to potential applications in tissue engineering and regenerative medicine (27-29). Commonly printed materials with biological additives (e.g., cells, enzymes, drugs) include alginate, agar and gelatin. These inks are typically prepared in liquid form and once printed, rapidly undergo a sol-gel phase transition facilitated by either physical or chemical crosslinking to form hydrogel structures (28). For hydrogel 3D printing, the development of inks is a challenging task. A proper ink for 3D printing has to satisfy many criteria, including: 1) sufficient viscosity to maintain the 3D shapes of printed filaments; 2) rapid gelation to minimize 11 ACS Paragon Plus Environment


spreading (on dry substrates) or diffusion (in solution) of printed filaments, but not too fast to result in clogging of the nozzle; and 3) sufficient mechanical stiffness after gelation to prevent collapse of the printed structure. Silk fibroin materials have been frequently used in 3D printing for tissue engineering (28). Silk and polyol aqueous-based inks allowed room temperature self-curing without the use of the ultraviolet (UV) irradiation or chemical crosslinkers, thus suitable for the incorporation of living cells and biomolecules (30). In a similar study, silk and gelatin-based 3D printing inks for engineering human nasal tissues was reported (31). Nasal mesenchymal progenitor cells were encapsulated in the 3D printed structures and survived the gelation process. In another study, silk-based 3D printing ink using gelatin as a thickening agent and physical crosslinking induced by glycerol generated printed structures with stability and tunable mechanics (32). 3D printing of self-standing structures using silk-based ink physically crosslinked by polyethylene glycol, exhibited cell compatibility (24). A photo-crosslinkable silk ink using methacrylation was used with digital light processing 3D printing for the construction of tissue constructs, such as heart, brain, trachea and ear (33). A typical approach to increase ink viscosity is to add a thickening agent, such as gelatin or agar (30, 32). However, these materials require elevated temperature to melt, which may adversely affect the stability of silk fibroin solutions (34, 35). Other biomacromolecules are also used, including cellulose/starch derivatives, carrageenan and guar gum (36-39). These materials are either difficult to dissolve in silk solution or are ionized in aqueous solution. The presence of high ion concentration in silk solution lowers the crosslinking efficiency (40, 41).


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Figure 1. Characterization of silk/HPMC hydrogels. (A) Dynamic viscosity of silk/HPMC ink mixtures at various silk to HPMC ratios. (B) The gelation kinetics of silk/HPMC ink mixtures with various silk-to-HPMC ratios and 180 unit/ml HRP (mean +/- SD, n=3). (C) Times to reach 50, 60, 70, 80 and 90% gelation for each silk-to-HPMC ratio with 180 unit/ml HRP (mean +/SD, n=3). (D) The Young’s modulus of silk/HPMC hydrogel at various silk to HPMC ratios and HRP concentrations after overnight crosslinking (mean +/- SD, n=2). (E) The optical transmittance of silk/HPMC hydrogel at various silk to HPMC ratios and 180 unit/ml HPR concentration. The absorbance peaks for chlorophyll A (430 and 660 nm) and B (450 and 640 nm) were highlighted on the graph. 13 ACS Paragon Plus Environment


Therefore, when choosing a thickening agent for silk hydrogel printing, the following criteria were considered: 1) room-temperature solubility; 2) easy dissolution in silk fibroin solution; and 3) absence of ionization in aqueous solution. Based on these criteria, hydroxypropyl methylcellulose (HPMC) was selected, which is chemically inert and biocompatible (42, 43). We tested the dynamic viscosity of ink mixtures at several different silk to HPMC ratios (Figure 1A). In the lower range of shear rate, all ink mixtures showed shear thinning behavior (44). At higher shear rates, sudden increases and then drops of viscosity occurred, especially at higher silk content mixtures. Previous studies from us and other groups showed shear thickening at higher shear rates, likely due to shear-induced crystallization as beta sheets (44-46). This was further confirmed by the observation that after the viscosity testing, the silk/HPMC ink samples became more opaque. According to these studies, the drop in viscosity following shear thickening may be due to precipitation of the beta sheet structures (45). The overall viscosity profile showed a linear relationship with HPMC content, where increasing HPMC content led to increased viscosity. The dynamic viscosity of pure silk and HPMC solutions at the same concentration as in the ink mixtures is shown in sFigure 1. The viscosity of pure silk and HPMC solutions was lower than the mixtures at all shear rates tested, suggesting interactions between silk fibroin and HPMC. Hydrogen bonds can form between the amine groups in the silk fibroin and the hydroxyl groups of HPMC, which are likely responsible for the increased viscosity of the silk/HPMC mixtures compared to the pure solutions of silk and HPMC (47-49). These ink mixtures were later tested in 3D printing to determine which viscosity was sufficient to maintain the 3D shapes of printed filaments.


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Next, we studied the gelation kinetics of the ink mixtures with different silk to HPMC ratios and HRP concentrations. It was reported that there was a positive correlation between silk fibroin gelation rate and HRP concentration (50), however the impact of HPMC content on the reaction kinetics was not clear. By examining the formation of phenolic crosslinking, with intrinsic fluorescence at 415nm when excited at 315nm, the gelation kinetics were quantified (Figure 1B, sFigure 2) (51). The gelation curves for each silk-to-HPMC ratio did not change significantly with changing HRP concentration (sFigure 2), suggesting that the gelation rate was limited by the diffusion of H2O2, not the HRP concentration. Across different silk-to-HPMC ratios, ink mixtures with higher silk content tended to gel faster (Figure 1B). The times to reach 50%, 60%, 70%, 80% and 90% gelation for the different ink mixtures are summarized in Figure 1C. The ink mixture with a 6:4 silk-to-HPMC ratio had the fastest gelation rate compared to the other ink mixtures. These ink mixtures were tested by printing to determine which silk to HPMC ratio was most suitable to balance ink viscosity with gelation rate. The addition of HPMC also influenced the Young’s modulus of the hydrogel (Figure 1D). Hydrogels printed with inks with higher silk contents tended to have higher mechanical strength. All ink mixtures with a 3:7 silk-to-HPMC ratio and ink mixtures with 4:6 silk-to-HPMC ratio and 60 unit/ml HRP did not form strong enough hydrogels to be tested, even after the overnight reactions, presumably due to low silk and HRP concentrations in the ink mixture. HRP concentration also had a positive impact on the final Young’s modulus. Higher Young’s modulus of final hydrogel was desired to enhance the mechanical stability of the printed structures. For the printing, we balanced ink viscosity with hydrogel mechanical strength to select the best ink formula for the 3D printing.



For microalgae immobilization, optical transparency is a key characteristic of the hydrogel material, as it directly affects light transmission and photosynthetic efficiency. Ink mixtures with different silk-to-HPMC ratios were printed in H2O2 solution and allowed to fully crosslink overnight. As can be seen in Figure 1E, the addition of HPMC had a significant impact on hydrogel optical transmission. Among the ink mixtures tested, the 6:4 silk to HPMC ratio provided the best optical transparency with higher than 60% transmittance at the absorbance peaks for chlorophyll A and B, the major chlorophylls in microalgae.

Figure 2. 3D printing of silk/HPMC ink mixtures. (A) A schematic illustrating the printing process of silk/HPMC ink mixture containing microalgae. (B) 3D printed structures (a squarebased pyramid and a bar spanning two conical shaped pillars) using the ink mixture with a 6:4 silk-to-HPMC ratio and 180 unit/ml HRP. The insets showed the 3ds MAX designs. Bars showed 1 cm.


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All ink mixtures formulated were tested for printability (Figure 2A). The ink mixture with a 6:4 silk-to-HPMC ratio and 180 unit/ml HRP generated printing outcomes that met the goals of the study: balancing viscosity, reaction kinetics and mechanics. The viscosity was sufficiently high to allow the filament to maintain its 3D shape and a 0.5 mm layer height. The gelation rate was sufficiently rapid that the printed structures could be properly crosslinked with good consistency to maintain structural integrity during long-term incubation. Premature gelation was not observed within the nozzle, thus it remained free of clogging during the entire printing process. 3D structures were successfully printed with the ink mixture with a 6:4 silk-to-HPMC ratio and 180 unit/ml HRP, such as a square-based pyramid and a bar spanning two conical shaped pillars to show the capacity of this silk/HPMC ink (Figure 2B). The ink mixture used for the printing of these demonstrative 3D structures did not contain microalgae and was dyed with food coloring for enhanced contrast.



Figure 3. 3D printing and characterization of microalgal silk hydrogel structures. (A) A 3D printed microalgal “tree” consisting four layers (2mm thick), along with an inset showing the 3ds MAX design. Bar showed 1 cm. (B) Left panel: phase contrast microscopic images of encapsulated microalgae at different time points showing long-term proliferation of microalgae within silk hydrogel matrix. Black arrows highlighted dividing microalgal cells. Bar showed 10 µm. Right panel: average daily increase of cell number at different time points (mean +/- SD, n=2, three images per sample per time point were analyzed). (C) Dissolved oxygen concentration measured before and after each medium change over a 90-day period showing long-term photosynthetic activity of encapsulated microalgae (mean +/- SD, n=2).

As a signature advantage of this new system, the silk hydrogel is able to provide a cell-friendly matrix that allows 3D encapsulation while maintaining normal cell proliferation and functions. We previously demonstrated 3D encapsulation of human fibroblast cells and mesenchymal stem cells within silk hydrogel matrices (15, 16). Here, we wanted to determine whether the silk hydrogels could also support long-term 3D cultures of the microalgae and maintain photosynthetic activity. We added microalgae in the silk/HPMC ink mixture with a 6:4 silk-toHPMC ratio right before printing. After the printed structures were completely crosslinked, the H2O2 containing medium was replaced with fresh microalgal medium to support the survival and proliferation of the microalgae (Figure 3A). Microalgal proliferation was determined by counting cell number increases under phase contrast microscopy for four weeks post printing (Figure 3B). The encapsulated microalgae showed rapid proliferation at more than 10% per day for up to 10 days after printing, after which proliferation slowed and reached more steady state; consistent with general growth curves of the microalgae used in our study (52, 53). The microalgae were 18 ACS Paragon Plus Environment

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not able to move within the hydrogel matrix due to the enzymatically crosslinked silk matrix. However, the cells were able to divide and maintained their normal elliptical shape of 3 to 4 µm in length and 2 to 3 µm in width (Figure 3B). This may be considered advantageous for certain environmental applications to avoid release of the algae into the specific environment. Using a non-invasive optical sensor, the photosynthetic activity of the 3D printed microalgal silk hydrogel structures was determined. The microalgal silk structures were immersed in microalgal medium, which was changed every three days and the oxygen concentration was measured before and after each medium change for 90 days. The test was conducted in BOD bottles with water seals to prevent gas exchange with the outside environment. A consistent 3 to 8 mg/L oxygen concentration increase was observed for every three-day period, supporting the stability and long-term function of these microalgal silk hydrogel structures (Figure 3C). The oxygen production of the microalgal silk hydrogel was slightly lower than free microalgae with the same cell number (sFigure 3, positive control), likely due to that fact that not all microalgae encapsulated in silk/HPMC hydrogel were able to exchange O2/CO2 with the medium due to diffusion limitations. As expected, the negative control (microalgae-free silk/HPMC hydrogels) did not produce oxygen (sFigure 3). The 3D printed microalgal silk hydrogel structures were stable during the 90-day photosynthesis experiment with no disintegration of the hydrogel structure observed. The gradual stiffening of silk hydrogel in salt-containing aqueous media (16, 17, 51) and the HPMC-silk interactions (47-49) likely contributed to this long-term stability. This stability and long-term activity suggest that the system may be suitable for environmental applications such as indoor air improvements and CO2 reduction with minimal human intervention.



One advantage of our system is that it presents minimal toxicity to the environment and to the human body because it involves naturally occurring materials (silk and microalgae) or modified natural materials (HPMC) that are processed (printing, gelation) in a safe, aqueous, room temperature process. Moreover, silk hydrogel materials can be degraded by proteases, such as protease XIV and proteinase K, and the end products are low molecular weight peptides (18). This enables our systems to be disintegrated and recycled for other uses, such as composting and generating biofuels after they complete their tasks. For indoor applications, safety is a top concern. As aforementioned, our systems do not involve any toxic chemicals. All materials are both bio-compatible and even edible; there are numerous consumer products sold that are ingested or applied to the body. Silk has also been used to coat perishable food to extend shelf life (54), and is used in medical products as degradable systems (13, 55). The consumption of microalgae as food and supplement also has a long history (56). Taken together, these unique advantages make our systems suitable for indoor and home utilities.

Conclusions A microalgae/silk ink with mechanical properties and gelation kinetics useful for 3D printing was developed and successfully utilized to host microalgae. The silk hydrogels provided a host environment to support the long-term proliferation and photosynthetic activity of encapsulated microalgae. Microalgae proliferation was demonstrated for more than 4 weeks and stable photosynthetic activity was observed for at least 90 days. The printability, stability and long-term functionality of such material supports potential environmental utilities.


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Supporting Information Available The following files are available free of charge: Supplemental Figures (PDF)

Acknowledgments We thank the NIH (P41 EB002520) for support of this work. The authors thank Dr. Maria Rodriguez and Dr. Nicole Raia for assistance with viscosity measurement, Dr. Nicole Raia for assistance with gelation kinetics measurement, and Dr. Wenyi Li for assistance with optical transmittance measurement and 3D printing.

Author contributions S.Z., F.G.O. and D.L.K. contributed to the initial concept. S.Z. and D.L.K designed the experiments. S.Z. and A.K. performed the materials optimization and 3D printing. C.G. performed the viscosity and Young’s modulus measurement of silk/HPMC hydrogel materials. S.Z. performed the long-term tests on microalgal proliferation and photosynthesis. S.Z. and D.L.K. wrote the manuscript, and all authors commented on the manuscript.

Conflict of Interest The authors declare no conflict of interest.



Data Availability The materials and protocol used, and the data generated in this study are available on reasonable request.

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3D printing of functional microalgal silk structures for environmental applications Siwei Zhao, Chengchen Guo, Allison Kumarasena, Fiorenzo G. Omenetto and David L. Kaplan


3D Printing of Functional Microalgal Silk Structures for Environmental

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