Glycerol

The frozen cell pellets prepared above were resuspended in 3 ml of 2 M NaOH (Fisher Scientific, Waltham, MA, USA) with 10% (vol/vol) beta-mercaptoethanol (Sigma-Aldrich, St. Louis, MO, USA). The solution was gently mixed and incubated on ice for 5 min to promote hydrolysis of the fungal cell wall. The solution was then centrifuged at 14,000 × g for 30 s at 4°C. The resulting pellet was resuspended in 3 ml of high-salt extraction buffer containing 40 mM HEPES, pH 7.5 (Fisher Scientific, Waltham, MA, USA), 350 mM NaCl (Fisher Scientific, Waltham, MA, USA), 0.1% (wt/vol) Tween 20 (Bio-Rad, Hercules, CA, USA), and 10% (vol/vol) glycerol (Fisher Scientific, Waltham, MA, USA). The solution was immediately centrifuged at 14,000 × g for 30 s at 4°C. The cell pellets were resuspended in 3 ml of 2× SDS sample buffer, 0.1 M Tris-HCl, pH 6.8 (Fisher Scientific, Waltham MA, USA), 4% (wt/vol) SDS (Fisher Scientific, Waltham, MA, USA), 0.2% (wt/vol) bromophenol blue (Sigma-Aldrich, St. Louis, MO, USA), 20% (vol/vol) glycerol (Fisher Scientific, Waltham, MA, USA), and 10% (vol/vol) beta-mercaptoethanol (Sigma-Aldrich, St. Louis, MO, USA). Cell pellets incubated at 100°C for 10 min, prior to being centrifuged at 14,000 × g for 30 s at 4°C. The supernatants were stored at −20°C until further use.

Water/glycerol mixtures were prepared by mixing glycerol (99.5%, Alfa Aesar) in ultra-pure water. A large range of concentrations, [0-99] wt.% with the range of viscosity, [1-900] mPa.s were investigated. The stock solution of 4-daspi (from Sigma-Aldrich) is prepared at ${10}^{-3}$  M in ultrapure water (with a resistance of 18.2 M $\mathrm{\Omega }$ ). The concentration of 4-daspi in water/glycerol solutions is ${10}^{-7}$  M to avoid dye-dye interactions.
All measurements were done with #1.5H coverslips (Deckgläser, thickness of $170\pm 5$   $\mathrm{\mu }$ m). The hydrophilic surfaces were prepared as follows. Coverslips were washed with ethanol (purity 99%), thoroughly rinsed in ultrapure water, dried in nitrogen and placed in a plasma cleaner for 30s. Hydrophobic surfaces were obtained by soaking the above coverslips for 15 min in a toluene/trichlorooctylsilane mixture (1%vol), rinsing with isopropanol and drying under nitrogen. Prepared glass was used immediately. The viscosity of the glycerol/water solution was measured by Anton-Paar MCR 302 rheometer.

Hydrogenolysis of glycerol to diols/alcohols was carried out with a 600 mL stirred reactor made of stainless steel (supplied by Parr Instruments Co., Moline, IL, USA). In a typical run, 18.5 g (0.2 gmol) glycerol (Alfa Aesar, Ward Hill, MA, USA), 0.63 g Re-Ru@SiO₂ nanoparticles prepared above, and 27.8 g water (i.e., 40 wt % glycerol aqueous solution) were mixed together and charged into the reactor. The agitator speed was set at 500 rpm, hydrogen was introduced into the reactor at a desired pressure, and the reaction mixture was then heated to the desired temperature. At the end of the reaction, the component compositions were determined with a Shimadzu (Kyoto, Japan) high performance liquid chromatography (model: LC-10A) equipped with a 250 mm long C-18 column and a UV detector (wavelength was set at 254 nm). The glycerol conversion was defined as (moles of glycerol reacted)/(moles of glycerol fed to the reactor) × 100%, product yield was defined as(moles of product obtained)/(moles of glycerol fed to the reactor) × 100%, product selectivity was defined as (moles of product obtained)/( moles of glycerol reacted ) × 100%.

Total cell lysates were prepared in a buffer containing 50 mM Tris-HCl (pH 7.8; Thermo Fisher Scientific), 137 mM NaCl (Thermo Fisher Scientific), 10 mM NaF (Thermo Fisher Scientific), 1 mM EDTA (Thermo Fisher Scientific), 1% Triton X-100 (Thermo Fisher Scientific), 10% glycerol (Thermo Fisher Scientific), and the protease inhibitor cocktail (Roche) through 3 freeze/thaw cycles. Tissue lysates were prepared by homogenizing in a buffer containing 50 mM Tris (pH 7.6; Thermo Fisher Scientific), 130 mM NaCl (Thermo Fisher Scientific), 5 mM NaF (Thermo Fisher Scientific), 25 mM β-glycerophosphoate (Thermo Fisher Scientific), 1 mM sodium orthovanadate (Thermo Fisher Scientific), 10% glycerol (Thermo Fisher Scientific), 1% Triton X-100 (Thermo Fisher Scientific), 1 mM dithiothreitol (Thermo Fisher Scientific), 1 mM phenylmethanesulfonyl fluoride (PMSF) (Thermo Fisher Scientific), and the protease inhibitor cocktail (Roche). After centrifugation (12,000g, 4°C for 10 minutes), tissue lysates were separated on SDS-polyacrylamide gel (SDS-PAGE) and analyzed using the following antibodies: rabbit anti-UCP1 (UCP11-A, Alpha Diagnostic), rabbit anti-HSP90 (sc-7947, Santa Cruz Biotechnology Inc.), rabbit anti–phospho-PKA substrate (9624, Cell Signaling Technology), rabbit anti-TH (ab112, Abcam), and mouse anti-tubulin (sc-32293, Santa Cruz Biotechnology Inc.).

In the printing process, three components were used to print the muscle constructs: the cell-laden bioink, the sacrificing acellular bioink, and the supporting PCL pillar. The cell-laden bioink composed of 20 mg/ml fibrinogen (Sigma), 35 mg/ml gelatin (Sigma), 3 mg/ml hyaluronic acid (HA, Sigma) and 10% (v/v) glycerol (Sigma) was prepared. Briefly, HA was dissolved in DMEM/high glucose at 37 °C with stirring overnight, and then glycerol was added to HA solution and stirred for 1 h. fibrinogen and gelatin were added to the HA/glycerol solution by gentle shaking at 37 °C for 1 h. After dissolution, the solution was sterilized by a 0.45 µm syringe filter (Thermo Scientific). Lastly, the cells were mixed with the bioink by gentle pipetting. The sacrificing bioink was prepared by dissolving 35 mg/ml gelatin, 3 mg/ml HA, and 10% (v/v) glycerol in DMEM/high glucose, and then filtered. PCL (Mw; 43,000~50,000, Polysciences, Inc., Warrington, PA) was used for the polymeric pillar structure.

E. coli MG1655 [28 (link)] was used as a parental strain, and the strains derived from the parental strain are mentioned in Table 1. All E. coli strains were maintained at 4 °C on LB agar plates supplemented with the required antibiotic. For all the quantitative measurements, the cells were first grown overnight in LB broth. A fraction of the cells were transferred to a fresh M9 salt medium supplemented with 0.1% glycerol (Affymetrix) and grown for 8 h at 37 °C in an incubator shaker. Later, a fraction of the cells were transferred to the required media for either growth measurements or fluorescence measurements. All experiments were performed in three biological replicates unless otherwise indicated.