Trichormus variabilis (Cyanobacteria) Biomass: From the Nutraceutical Products to Novel EPS-Cell/Protein Carrier Systems

Abstract

A native strain of the heterocytous cyanobacterium Trichormus variabilis VRUC 168 was mass cultivated in a low-cost photobioreactor for a combined production of Polyunsaturated Fatty Acids (PUFA) and Exopolymeric Substances (EPS) from the same cyanobacterial biomass. A sequential extraction protocol was optimized leading to high yields of Released EPS (REPS) and PUFA, useful for nutraceutical products and biomaterials. REPS were extracted and characterized by chemical staining, Reversed Phase-High-Performance Liquid Chromatography (RP-HPLC), Fourier Transform Infrared Spectroscopy (FT-IR) and other spectroscopic techniques. Due to their gelation property, REPS were used to produce a photo-polymerizable hybrid hydrogel (REPS-Hy) with addition of polyethylene glycol diacrylated (PEGDa). REPS-Hy was stable over time and resistant to dehydration and spontaneous hydrolysis. The rheological and functional properties of REPS-Hy were studied. The enzyme carrier ability of REPS-Hy was assessed using the detoxification enzyme thiosulfate:cyanide sulfur transferase (TST), suggesting the possibility to use REPS-Hy as an enzymatic hydrogel system. Finally, REPS-Hy was used as a scaffold for culturing human mesenchymal stem cells (hMSCs). The cell seeding onto the REPS-Hy and the cell embedding into 3D-REPS-Hy demonstrated a scaffolding property of REPS-Hy with non-cytotoxic effect, suggesting potential applications of cyanobacteria REPS for producing enzyme- and cell-carrier systems.

Keywords: Cyanobacteria; Extracellular Polymeric Substances; PUFA; Trichormus variabilis; biomaterials; enzyme; hydrogel; mesenchymal stem cells; omega 3.

Figure 1

Biomass production. Light micrograph of T. variabilis trichomes in: culture (A); bench-scale growth system used (B,C); and pilot-scale growth system used (D).

Figure 2

Growth curve recorded in polyethylene bag growth experiment (A). Light micrographs of cultures after Alcian Blue staining at pH 0.5 (B) and 2.5 (C) for sulfated and carboxylic EPS residues, respectively. Sulfated EPS were diffluent and less abundant (B), while carboxylic groups were observed in more bound matrix material (C).

Figure 3

Characterization of REPS: (A) RP-HPLC chromatogram of REPS solution (10 mg/mL) using C₁₈ column at 0.8 mL/min flow rate and the following gradient: 0–5 min, 0%; 5–50 min, 60%; 50–55 min, 60%; 55–60 min, 90%; and 60–65 min, 90% of solvent B (80% v/v CH₃CN and 0.1% v/v TFA). Inset: UV-vis absorption spectrum of REPS solution. (B) FT-IR spectrum of REPS showing signals within 4000 to 250 cm⁻¹; the measurements were consistent among three replicates.

Figure 4

Synthesis and characterization of the REPS-Hy: (A) Schematic representation of REPS-Hy production: the gelling of the solution of 8.83 mg/mL of REPS with 3% of PEGDa (6 kDa) (w/v), and 0.1% of Irgacure^®2959 (w/v) was obtained after 5 min of UV light (365 nm) exposition. (B) Resistance of REPS-Hy to dehydration and spontaneous hydrolysis. Digital macro-photographs of: PEGDa-Hy with 2% of PEGDa without REPS and REPS-Hy with 2% of PEGDa (w/v) (top); REPS-Hy with 3% of PEGDa-Hy (w/v) at Day 0 and Day 14 after storage at 4 °C, and after 72 h of incubation at 37 °C in PBS (bottom). (C) Swelling rate curves of REPS-Hy and PEGDa-Hy in PBS up to the equilibrium swelling (30 h). The R² of the hyperbolic fits of the swelling trend of REPS-Hy and PEGDa-Hy are 0.9284 and 0.9912, respectively.

Figure 5

REPS improve hydrogel mechanical properties. Rheological measurement of REPS-Hy and PEGDa-Hy as evaluated by: time-sweep tests (A); frequency-sweep tests (B); and strain-sweep tests (C). The shear storage modulus (G′) and shear loss modulus (G′′) are shown for both hydrogels. (A) The time sweep data reveal an increase in G′ upon the light-activated free-radical polymerization reaction of the REPS-Hy and PEGDa-Hy liquid precursors. The plateau G’ of the REPS-Hy was 55% higher as compared to the plateau G′ of the PEGDa-Hy, indicating that the REPS improves the elastic mechanical properties of the hydrogels. Following the chemical cross-linking of the hydrogels, the frequency-sweep (B) and strain-sweep (C) data confirmed a linear relationship between the shear modulus in the range of the applied frequency and strain.

Figure 6

REPS-Hy as detoxification enzyme-encapsulating hydrogel: (A) Scheme of the catalytic cycle of the TST enzyme. (B) Percentage of TST activity over time of 3.12 μM of TST in presence of 50 μL of PBS (white) or of REPS solution (8 mg/mL of REPS and 8 mM of CaCl₂ in PBS, pH 7.4) (black) (100% is the activity in PBS at time 0). The Sörbo assay was performed at 23 °C. (C) TST activity of TSTREPS-Hy at different incubation times (5, 15, 30 and 60 min) at 37 °C. The reaction was stopped after incubation and the absorbance of the solutions measured after dilution. The line equation is y = 0.07611x and R² is 0.9621. (D) TST activity of TSTREPS-Hy at time 0 and after 20 h at room temperature (23 °C) in 200 µL of 50 mM Tris-HCl, pH 8.0, buffer (100% is the TSTREPS-Hy activity at time 0). *** p < 0.001, n = 3 or 5.

Figure 7

hMSCs cultures using REPS-Hy as scaffold. Confocal laser scanning micrographs of hMSCs cultured for two weeks on REPS-Hy: (A) at 60× magnificantion; (B) at 40× magnification; and (C) micrographs with Y–Z and X–Z projections. F-actin was stained with Alexa-fluor 568 phalloidin-conjugate (in red) and the nuclei were stained using Hoechst 33342 (in blue). Scale bars = 10 μm.

Figure 8

REPS-Hy as stem cell-carrier system: (A) Schematic representation of the production of REPS-Hy scaffolds for 3D hMSC cultures. (B) Cell viability of hMSCs embedded into the REPS-Hy, immediately after photo-polymerization (time 0) and after 72 h of 3D cell culture (100% is the cell viability at time 0). (C) Digital macro-photographs of REPS-Hy with and without embedded hMSCs after colorimetric WST-1 cell viability assay. *** p < 0.001.