Biomate 5

M. smegmatis cells were grown in standard Middlebrook 7H9 medium (BD/Difco) containing 0.085% NaCl, 0.5% albumin, 0.2% glucose, 0.5% glycerol, and 0.05% Tween-80 at 37 °C with shaking at 200 rpm [35 (link)]. Optical density of the cells (absorbance at 600 nm) was measured to determine the growth of the cells via a spectrophotometer (Thermo Scientific biomate 5, Waltham, MA, USA).

Cell concentration was monitored by measuring OD at 600 nm using a UV-visible spectrophotometer (Biomate5, Thermo, USA). Glucose, cellobiose, cellodextrin and ethanol concentrations were determined by high performance liquid chromatography (HPLC; Agilent Technologies 1200 Series, Agilent, USA) equipped with a refractive index (RI) detector using a Rezex ROA-Organic Acid H⁺ (8%) column (Phenomenex Inc., USA). The column was eluted with 0.005 N of H₂SO₄ at a flow rate of 0.6 ml/min at 50°C.
Extracellular β-glucosidase activity was measured according to the methods reported previously [20 (link)-22 (link, link)]. The culture broth was harvested and centrifuged at 15,000 ×g for 20 min at 4°C. After filtration of the supernatant with a 0.22-μm spin filter, the filtrate (500 μl) was mixed with the same volume of 50 mM sodium citrate buffer (pH 4.8). After addition of 6.7 mM p-nitrophenyl-β-D-glucopyranoside (p-NPG), release of p-nitrophenol (p-NP) was monitored by a spectrophotometer at 400 nm. One unit (U) of β-glucosidase was defined as the amount of enzyme that catalyzes the release of 1 μmol of p-NP per min at 30°C. Volumetric extracellular β-glucosidase activity was expressed as units of β-glucosidase per liter. Specific extracellular β-glucosidase activity was expressed as units of β-glucosidase per gram of dry cell.

Surface analyses were performed by soaking an MS plate with dimensions of 1 × 1 × 0.1 cm³ for 6 h in acid solution without DPH and with a 1000 ppm DPH. After that, it was rinsed with deionized water and dried at 60 °C in an oven. FE-SEM (Mira (III) TE-Scan) with a 15 kV field emission source (Oxford Instruments EDX Microanalysis X-MAX-80) were utilized to examine the morphology and elements on the MS surface. AFM (BRUKER, ICON, United States) was used to study the surface topology. X-ray photoelectron spectroscopy (XPS, BESTEC EA10, Germany), grazing incidence X-ray diffraction (GIXRD, PHILIPS, PW1730, Netherlands), and Fourier-transform infrared spectroscopy (FTIR, THERMO, AVATAR, United States) analyses were also used to investigate the surface layer composition formed on MS. The adsorption of DPH on the metal surface was studied using ultraviolet–visible spectroscopy (UV–Vis, THERMO, BIOMATE5, United States). Hydrophobicity/hydrophilicity and surface chemistry of MS surface was also explored using a contact angle device.

A spectrophotometer (Thermo Scientific biomate 5) was used to determine turbidity of cultures by measuring optical density (absorbance at 600 nm), using 1 ml cuvettes. Dilutions were made in 7H9, to keep the reading in the linear range of the spectrophotometer.

The OD₆₀₀ of cultures was measured using a spectrophotometer (Biomate 5, Thermo, NY) and dried cell weight (DCW) was obtained from an experimentally determined conversion factor of 0.454 g DCW/OD₆₀₀. Extracellular metabolites such as lactose, galactose, galactitol, glycerol, acetate, and ethanol were measured by HPLC (Agilent Technologies 1200 Series, Santa Clara, CA) with a Rezex^TM ROA-Organic Acid H + (8%) column (Phenomenex Inc., Torrance, CA) and a refractive index detector (RID). The column was eluted with 0.005 N H₂SO₄ at a flow rate of 0.6 mL/min at 50 °C. Galactose and tagatose could not be separated by this ROA-organic acid H + (8%) column (Supplementary Figure 3). Galactose and tagatose concentrations were measured using an Agilent Technologies 1200 Series HPLC equipped with a Rezex^TM RCM-Monosaccharide Ca + 2 (8%) column (Phenomenex Inc. Torrance, CA) and RID detector. The mobile phase was E-Pure™ Water (Barnstead E-Pure™ Water Purification Systems, Thermo Scientific) and was eluted at a flow rate of 0.6 mL/min at 80 °C. The maxium theoretical tagatose yield (0.526 g tagatose/g lactose) by engineered yeast was calculated based on the molecular weights of tagatose (180.16 g/mol) and lactose (342.3 g/mol) with an assumption that glucose is not converted into tagatose.

S. cerevisiae strain EJ4 capable of efficient xylose and cellobiose fermentation [7 (link)] was cultivated in yeast synthetic complete broth containing 6.7 g/L yeast nitrogen base, 0.79 g/L complete supplement mixture (CSM; MP Biomedicals, Solon, OH), and 20 g/L carbon source such as glucose, galactose, xylose, or cellobiose at 30 °C and 200 rpm. Culture samples were collected in the exponential phase of growth for metabolite profiling. Cell growth was monitored by measurement of optical density at 600 nm (OD₆₀₀) using a UV–visible spectrophotometer (Biomate5, Thermo Fisher Scientific, Waltham, MA).