U 1100 spectrophotometer

Fluorescent conjugates were constructed using Alexa Fluor 488 5-TFP (Life Technologies, Eugene, OR, USA) following the manufacturers protocol and methodology previously described in [15 (link)]. Saporin SO6 or OKT10-SAPORIN were added to 100 µL carbonate buffer (1 M NaHCO₃, pH 9.0) and 100 µL of Alexa Fluor 488 5-TFP (10 mg/mL in DMSO) and stirred for one hour at room temperature. Unconjugated fluorophore was removed by dialysis against PBS at 4 °C. The Beer–Lambert law was used to determine the concentrations of the fluorescent conjugates by measuring the absorbance of the samples at 280 and 495 nm using a Hitachi U1100 Spectrophotometer.

Headspace samples were analyzed for CO₂, H₂, and CH₄ by gas chromatography, using a dual channel Micro-GC (CP-4900; Varian, Micro gas chromatography, Middelburg, The Netherlands), as previously described [8 (link)]. The results were analyzed with a Galaxie Chromatography Workstation (version 1.9.3.2, Middelburg, The Netherlands). The optical density of the culture was measured at 620 nm (OD₆₂₀) using a U-1100 spectrophotometer (Hitachi, Tokyo, Japan). CDW was determined by filtration as previously described [24 (link)]. Glucose, acetate, lactate, propionate, and ethanol were analyzed by HPLC (Waters, Milford, Massachusetts, United States) on an Aminex HPX-87H ion exchange column (Bio-Rad, Hercules, United States) at 45°C, with 5 mM H₂SO₄ (0.6 ml · min⁻¹) as the mobile phase. The column was equipped with a refractive index detector (RID-6A; Shimadzu, Kyoto, Japan).

The protein concentration of mitochondrial preparations was determined by the Biuret method (Lowry, Rosebrough, Farr, & Randall, 1951). The number of LtP was determined by optical density at 600 nm (HITACHI U‐1100 Spectrophotometer, Japan). The cell broth was diluted 1:10 with culture medium and measured against a blank of culture medium. The cell number was calculated using the formula (Fritsche, Sitz, Weiland, Breitling, & Pohl, 2007). Two replicates of each culture were performed.

The evaluation of color intensity was based on optical comparison using simple color grading as defined by Pfund and Lovibond [51 ,52 (link)]. In general, honey is marked according to the Pfund color scale, so Lovibond graders on a Pfund scale are used. Other more objective methods have also been used, such as the determination of all color parameters by the CIELAB L*a*b* three-dimensional method [52 (link),53 ,54 ]. The CIELAB system is a reflection method (measuring geometry d80, illuminant D65, range 400–700 nm, observer 10°) performed on a Hitachi model U-1100 spectrophotometer (L* lightness, a* chromaticity +red/−green, b* chromaticity +yellow/−blue, C*ab chroma, hab, 10).

Nitrogen content (N_cont), Chlorophyll a (Chl a), Chlorophyll b (Chl b), and Chlorophyll a + b (Chl a + b) were quantified at the same leaves where the gas exchange was measured. For measuring Chl a, Chl b, and Chl a + b; four-leaf disks of 0.5 cm diameter were taken on 30 July and dissolved in 2 ml N, N-Dimethylformamide (DMF) as described by Porra (2002) (link). The samples were then wrapped in aluminum foil and stored at 5°C for over 24 h. Spectroscopic readings of the supernatant were taken at 646.8 nm, 663.8 nm, and 750 nm wavelengths using a U-2910 Spectrophotometer (HITACHI, Japan). Nitrogen content was measured on 30 July and 11 August. The leaf area was measured using a leaf area meter LI-3100C (LI-COR, United States). They were then oven-dried for 72 h and were used for quantifying N_cont using Kjeldahl digestion. We followed the method of Vickery (1946) (link) for quantifying N_cont. The oven-dried leaves were crushed, and a sample of 0.2 g was dissolved and heated in 4 ml of highly concentrated sulfuric acid until the solution turned colorless. The colorless solution was then diluted to 40 ml with distilled water. We separated 20 μl aliquots of this solution into glass tubes and mixed it with 2.48 ml of distilled water, 1 ml of Indophenol A, and 1.5 ml of Indophenol B. These were then read at 635 nm wavelength using a U-1100 Spectrophotometer (HITACHI, Japan).

The cell dry weight was measured by filtering known volumes of the cultures through pre-dried and pre-weighed 0.45-μm-pore size nitrocellulose filters. The filters with the biomass were washed with water, dried for 15 min in a microwave oven at 150 W and weighed again. The optical density at 600 nm was determined using a Hitachi U-1100 spectrophotometer. For the intracellular concentration estimation, the cell volume (V_C) was calculated to V_C=2.38 ml (g CDW)⁻¹ (ref. 57 (link)).