Sustained Photosynthesis and Oxygen Generation of Microalgae-Embedded Silk Fibroin Hydrogels

Microalgae immobilized in hydrogels offer advantages over those cultured in suspension culture in terms of carbon fixation and oxygen emission. However, alginate as a commonly used hydrogel for microalgal immobilization encounters problems with mechanical strength and stability. To address this limitation, silk fibroin (silk) hydrogels prepared by ultrasonication were utilized to host microalgae when mixed with the presonicated protein solution prior to its gelation. The gelation time, stability, and light transmission of these silk gels were evaluated, and a silk concentration of 4% w/v and a gel thickness of 1 mm provided mechanical strength and stability during algal culture in comparison to alginate hydrogels. Furthermore, silk hydrogels with algal cell densities of 7.6 × 105 and 7.8 × 107 cells/mL had better stability than those with a lower cell density (3.2 × 103 cells/mL), likely due to cell confinement and impact on proliferation. The silk hydrogels with microalgae at a high density generated 6.13 mg/L of oxygen continuously for 7 days. An oxygen-generating device was fabricated by coating the surface of a dialysis tube with a thin layer of the microalgae-embedded silk hydrogel, where the microalgal cells were nourished with a culture medium prefilled in the dialysis tube. When suspended in a sealed flask filled with CO2 gas, the system continuously produced oxygen (151 mL) for at least 60 days, with an oxygen production efficiency 6 times that of microalgal suspension culture controls. This microalgae embedding and cultivation technique could have potential utility in air purification, tissue repair, and other applications due to the efficient and sustained generation of oxygen.

Characterization of silk hydrogels.

(A) HRP gel dissolution: u.v. absorbance (280 nm) of silk in the culture medium (mean ± SD, n = 3). (B) Sonicated gel dissolution: u.v. absorbance (280 nm) of silk in culture medium (mean ± SD, n = 3). (C) Weight loss of sonicated gels and alginate gels after 10 days of immersion in culture medium (mean ± SD, n = 3); * indicates significant difference between samples (p < 0.05). (D) Light transmission of sonicated hydrogels. Gel thickness was 1, 2, and 3 mm, and the concentrations of silk used to prepare the hydrogels were 2, 4, and 6% (w/v). The dotted lines indicate the absorbance maxima of chlorophyll A (430 and 660 nm). (E) Photographs of three microalgae-embedded hydrogels (4% HRP gel, 4% sonicated gel, and 2% alginate gel, with cell densities of 8 × 107, 7.8 × 107, and 3.2 × 103 cells/mL, respectively).

Microalgae are the most primitive and largest autotrophic oxygen-producing organisms on earth, and 40% of the oxygen on the earth comes from their photosynthetic activity. Similarly, 50–80% of the earth’s oxygen comes from the ocean, most importantly from marine plankton-drifting plants, algae, and some bacteria that utilize photosynthesis. For example, Prochlorococcus is the smallest photosynthetic organism known on earth, yet produces 20% of the oxygen in the biosphere, higher than all of the rainforests on land combined. Microalgae have advantages of high photosynthetic efficiency, easy culture, short growth cycle, high oil content, and environmental adaptability. Due to these advantages, microalgae are considered to be suitable candidates for carbon fixation, wastewater treatment, hydrogen production, and biodiesel formation. Microalgae also contain unique primary or secondary metabolites, such as proteins, vitamins, and minerals, which can be used to produce healthy foods, consumer cosmetic products, and natural fertilizers, among others. Recently, microalgae have been used as a green source to generate oxygen to improve air quality, such as for offices and malls where the air (oxygen) needs to be refreshed frequently. Commonly used ventilation systems are energy-consuming and generate greenhouse gas (GHG) emissions. Green biotechnologies, such as the use of microalgae to convert CO2 to O2 through photosynthesis, would be an ideal solution for reducing GHG and improving air quality. In addition to environmental applications, microalgae have been used to promote wound healing and tissue regeneration, based on oxygen and metabolites, which are beneficial for cell proliferation and differentiation.

Characterization of microalgae proliferation in silk and alginate hydrogels.

(A) Flowchart of preparation of microalgae-embedded sonicated gels. (B) Chlorophyll fluorescence of microalgae with different inoculation densities in the sonicated gels (mean ± SD, n = 3). (C) Chlorophyll fluorescence of microalgae with different inoculation densities in the HRP gels (mean ± SD, n = 3). (D) Chlorophyll fluorescence of sonicated gels and alginate gels with the same cell inoculation density (3.2 × 103 cells/mL) (mean ± SD, n = 3). (E) Metabolic activity (MTT) of cells at different densities in sonicated gels (mean ± SD, n = 3). (F) Proliferation rate of cells at different densities in sonicated gels (mean ± SD, n = 3). (G) Photographs of microalgae-embedded sonicated gels with different cell densities: 3.2 × 103, 7.6 × 105, and 7.8 × 107 cells/mL. (H) Live/dead staining of microalgae in sonicated gels and alginate gels on day 10. H-1, H-2, H-3, H-5, H-6, and H-7 are sonicated gels, and H-4 and H-8 are alginate gels. The microalgae density in H-1/H-5 and H-4/H-8 was 3.2 × 103 cells/mL, while those in H-2/H-6 and H-3/H-7 was 7.6 × 105 and 7.8 × 107 cells/mL, respectively. Live cells emitted red fluorescence (chlorophyll A, excitation wavelength = 569 nm), while dead cells emitted green fluorescence (excitation wavelength = 488 nm). Scale bar = 50 μm; * and ** indicate significant difference between samples (p < 0.05, p < 0.01).

Although microalgae hold potential for a variety of applications, a number of challenges remain to develop the technology, including the small size of the microalgae (3–30 μm), which makes them difficult to collect, and the low yield of high value-added products in microalgae. These issues require significant expansion of cultures to obtain sufficient biomass, but this results in increased costs due to labor and material resources needed for handling the biomass. These problems are less significant when considering the utilization of microalgae for oxygen generation for air purification; however, the efficiency of oxygen generation is low for microalgae in suspension cultures. The most promising option to address this issue is to use immobilization technology to fix microalgae in a limited space, with retention of photosynthetic activity over an extended time frame, whereby utilization efficiency improves due to the repeated use. In addition, immobilized microalgae exhibit a higher photosynthetic efficiency compared to microalgae in suspension culture due to the differences of light transmission between solid materials (usually small particles or thin layers of hydrogel) and bulk solutions.

Photosynthesis of microalgae embedded in silk hydrogels compared to those suspended in culture medium.

(A) Photo-bioreactor used in the experiment. (B) Schematic of the photo-bioreactor with the polyester fabric coated with microalgae-embedded silk hydrogel floating in the culture medium. (C) Microscopic image of the polyester fabric coated with microalgae-embedded, sonicated silk hydrogel. (D) The concentration of dissolved oxygen in the culture medium generated by the microalgae embedded in sonicated gel and suspended in the culture medium (mean ± SD, n = 3). The initial oxygen was eliminated by Na2SO3 at the beginning of a test. (E) Oxygen production efficiency for the cell embedded gels and the suspended microalgae in the media (mean ± SD, n = 3); * and ** indicate a significant difference between samples (p < 0.05, p < 0.01).

There have been many studies on carrier materials suitable for microalgal immobilization. Nonmetallic materials, such as porous glass and porous porcelain, are commercially available and have been widely used, but the adsorption capacity of microalgae to these materials is low. Thus, surface modification is often needed to enhance the absorption and retention of microalgae. For synthetic polymers,such as polyethylene, polyacrylamide, and polyurethane, processing also involves the use of organic solvents that can limit utility for microalgal immobilization. Natural polymer materials (agar, alginate, collagen, carrageenan) are widely used carrier materials due to the aqueous and biocompatible mild processing conditions, with significant advantages including low cost, biocompatibility, biodegradability, and sufficient mechanical properties. In recent years, alginate gel is considered a promising biomaterial in tissue engineering. Gelation can be achieved after mixing the alginate solution with cation (calcium and barium) at room temperature, and cells can be embedded in the gel, and proliferate and exert tissue regenerative functions in the body post-implantation. A photo-bioreactor for immobilizing Rhine algae was reported using alginate gels, and hydrogen production efficiency in the reactor was assessed. The reactor produced hydrogen continuously for about 10 days, and 20 mL of hydrogen was accumulated per gram of microalgae during this period frame. However, alginate gels are relatively unstable over time and dissolve in cell culture due to the high concentration of cations (Mg2+, K+), resulting in cell leakage from the materials. To address this issue, liquid–gel transition gels (agar) and immobilized Chlorella were utilized; however, the temperature conditions for agar to form a gel must be above 37 °C, which may damage or kill microalgae, as the optimal temperature for the growth and survival of most microalgae is 25–30 °C.

Generation of gaseous oxygen by microalgae-embedded silk hydrogels.

(A) Schematic of the process to prepare silk/microalgae gel-coated dialysis tubes and to collect gaseous oxygen in 60 days. (B) Photographs of the gel-coated dialysis tube and the apparatus used to culture the gel system and to collect oxygen. (C) SEM images showing the microalgae/silk hydrogel attached to the semipermeable membrane C-1 and C-4 showing the pores of the sonicated gel, C-2 and C-5 showing the contact interface between the gel matrix and the membrane, and C-3 and C-6 showing the microalgal cells embedded in the gel matrix. (D) The total volume of gas collected in the collection flask (mean ± SD, n = 3). (E) Determination of gas composition in the sampling gas. (F) The method used to calculate carbon sequestration by the microalgae/silk hydrogel.

Silk fibroin protein (silk) is a natural protein-polymer purified from silkworm cocoons. Tissue engineering scaffolds and drug delivery carriers prepared from silk exhibit excellent biocompatibility, tunable rates of biodegradability, robust mechanical properties, low immunogenicity, and minimal inflammatory reactions. Silk can be prepared in various biomaterial formats, such as porous scaffolds, films, hydrogels, nanoparticles, nanofibers, and microspheres, using aqueous systems with physical cross-linking (thus avoiding harsh chemicals often required for chemical cross-linking or photo-cross-linking). Biomaterials prepared from silk support cell proliferation and differentiation. Applying ultrasonic conditions to silk solutions induces gelation through physical cross-linking between the hydrophobic domains of the protein, leading to the formation of β-sheet (crystalline) domains. The gelation process can be conducted under ambient conditions and gelation time can be adjusted from minutes to hours, with cells mixed with the presonicated solution prior to gelation, thus avoiding direct sonication impact on the cells. The sonicated silk hydrogel has good mechanical properties, with compressive moduli in the range of 369–1712 kPa, and silk hydrogels at 4% (w/v) were suitable for the encapsulation of human bone marrow-derived mesenchymal stem cells (hMSCs); cells retained viability and proliferation in static culture over weeks. Silk hydrogels can also be prepared under ambient conditions using chemical cues as needed to generate more robust gel matrices, such as with H2O2-horseradish peroxidase (HRP) reactions that induce silk cross-linking via di-tyrosine bonds. Based on this approach, a microalgae/silk hydrogel ink suitable for underwater three-dimensional (3D) printing was developed and used for oxygen supplementation and carbon dioxide reduction. Microalgae embedded in the silk-HRP hydrogel maintained activity with stable photosynthetic activity for at least 90 days.

In light of these results, the objective of the present work was to compare different types of silk hydrogels for microalgal immobilization, as well as different cell seeding and culture conditions. To enable microalgae-embedded silk hydrogels applicable for air purification, a prototype device was also developed using semipermeable membranes as the substrate to support a thin gel layer coated on the membrane surface and exposed to air. The results confirmed that microalgae embedded in the sonication-induced silk hydrogels exhibited slow proliferation with high and sustained photosynthetic activity, with the release of significant oxygen to the medium and air1.

  1. Yuhang Fu, Xusheng Xie, Yongfeng Wang, Jian Liu, Zhaozhu Zheng, David Lee Kaplan, and Xiaoqin Wang ACS Biomaterials Science & Engineering Article ASAP DOI: 10.1021/acsbiomaterials.1c00168

1 comment