To monitor growth, absorbance at 660 nm measurements was performed on a JENWAY 7200 spectrophotometer (Cole-Parmer). The biomass dry weight was determined in duplicate as described earlier (31 (link)). For extracellular metabolite determinations, broth samples were centrifuged for 5 min at 13,000g, and the supernatant was collected for analysis by HPLC using an Aminex HPX-87H ion exchange column (Agilent). The HPLC was operated at 60 °C, and 5 mM of H2SO4 was used as mobile phase at a rate of 0.6 ml/min. Off-gas concentrations of CO2 and O2 were measured using an NGA 2000 analyzer. Proteome samples (∼3–5 mg dry weight) were taken from batch cultures. The samples were collected in multifold in trichloroacetic acid (TCA) (Merck Sigma; catalog no.: T0699) with a final concentration of 10%. Samples were centrifuged at 4000g for 5 min at 4 °C. Cell pellets were frozen at −80 °C (32 (link))
Jenway 7200 spectrophotometer
Manufactured by Cole-Parmer
Sourced in United Kingdom
The JENWAY 7200 spectrophotometer is a digital instrument designed for the measurement of absorbance, transmittance, and concentration of samples in the UV-Vis spectrum. It features a wavelength range of 190 to 1100 nm and a photometric range of -0.3 to 3.0 Abs.
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7 protocols using jenway 7200 spectrophotometer
1
Biomass and Metabolite Monitoring in Batch Cultures
2
Yeast Growth and Carbon Source Utilization
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Cells from a frozen stock were inoculated in liquid YPM or SM medium supplemented with maltose, uracil, histidine, leucine and tryptophan and grown aerobically at 30°C, 200 rpm. After 8 h, the optical density at 660 nm was measured with a JENWAY 7200 spectrophotometer (Cole-Parmer, Stone, UK). An appropriate volume of cell suspension was spun down (3000 g, 2 min at 4°C) and washed with sterile water, then transferred to a fresh 500 mL shake flask with an initial OD660 of 0.5 and cultivated for 4 hours at 30°C. After measuring OD660 in triplicate, cells were collected by centrifugation (3000 g, 2 min at 4°C), washed with sterile water and resuspended to a concentration of 107 cells mL−1. From this cell suspension, a 10x serial dilution was prepared in water with final concentration of 103 cells mL−1. Then, 10 μL of each concentration were spotted on selective SM agar plates supplemented with uracil, tryptophan, leucine and histidine with 20 g L−1 glucose, 6.8 g L−1 maltose, 20 g L−1 mannose, 20 g L−1 galactose, or 20 g L−1 fructose as carbon source and grown for 2 days at 30°C.
3
Anaerobic Growth Rate Determination
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The growth rate of the constructed strains was determined in 500-mL shake flasks containing 100 mL of SMD or SME medium. Wake-up cultures were inoculated with a 1-mL aliquot of a strain stored at −80°C and grown until late exponential phase. Precultures were inoculated from the wake-up cultures and grown to mid-exponential phase. Finally, measuring cultures were inoculated in biological duplicates from the preculture at an initial optical density at 660 nm (OD660) of 0.3. Cultures were monitored based on the OD660 with a Jenway 7200 spectrophotometer in technical duplicate (Cole-Parmer, Vernon Hills, IL). A maximum specific growth rate (μmax) was calculated from at least five data points in the exponential phase with at least 2 doublings.
Anaerobic shake flask-based experiments were performed at 30°C in a Bactron anaerobic chamber (Sheldon Manufacturing Inc., Cornelius, OR) with an atmosphere of 5% (vol/vol) H2, 6% (vol/vol) CO2, and 89% (vol/vol) N2, on an IKA KS 260 basic shaker at 200 rpm, using 50-mL shake flasks containing 30 mL SMD (2%) anaerobic medium.
Anaerobic shake flask-based experiments were performed at 30°C in a Bactron anaerobic chamber (Sheldon Manufacturing Inc., Cornelius, OR) with an atmosphere of 5% (vol/vol) H2, 6% (vol/vol) CO2, and 89% (vol/vol) N2, on an IKA KS 260 basic shaker at 200 rpm, using 50-mL shake flasks containing 30 mL SMD (2%) anaerobic medium.
4
Bioreactor Cultivation Analysis Protocol
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Offgas analysis, biomass dry weight measurements, HPLC analysis of culture supernatants and correction for ethanol evaporation in bioreactor experiments were performed as previously described (Papapetridis et al.2016 (link)). Determination of optical density was performed at 660 nm using a Jenway 7200 spectrophotometer (Cole-Palmer, Staffordshire, UK). Yields of products and biomass-specific sugar uptake rates in bioreactor batch cultures were determined as previously described (Wisselink et al.2009 ; Papapetridis et al.2016 (link)). All values are represented as averages ± mean deviation of independent biological duplicate cultures.
5
Measuring Bacterial Growth Kinetics
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Optical density of aerobic cultures was measured at 660 nm (OD660) with a Jenway 7200 Spectrophotometer (Bibby Scientific, Staffordshire, UK). Maximum specific growth rates were calculated from the slope of ln OD660 versus time during the exponential phase, considering at least six time points. Optical density measurements in anaerobic cultures were performed at 600 nm with an Ultrospec 10 cell density meter (Biochrom, Harvard Biosience, Holliston MA) placed in the anaerobic workstation. For spot-plate experiments, cell counts in late-exponential-phase shake-flask cultures were first determined using a Z2 Coulter Counter Analyzer (Beckman Coulter Life Sciences, Indianapolis IN) following the manufacturer’s protocol. Prior to analysis, cultures were diluted one 100-fold with Isoton II (Beckman Coulter, Brea CA), followed by five replicate cell counts on each sample.
6
Quantifying Microbial Metabolites and Compounds
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Extracellular metabolite concentrations were analyzed by high-performance liquid chromatography (86 (link)). Extraction and quantification of fatty acids, sterols, and tetrahymanol by GC-FID was performed as previously described (20 (link), 36 (link)). A 6-point calibration of the GC-FID system with hop-22(29)-ene (Sigma-Aldrich, 0.1 mg ⋅ mL−1 in isooctane) was used for quantification of hop-22(29)-ene and other detected hopanoid compounds. Detailed methods for detection and identification of hopanoid compounds by GC-MS are described in SI Appendix . Optical density was measured at 600 nm for anaerobic cultures with an Ultrospec 10 cell density meter (Biochrom, Harvard Biosience) placed in the anaerobic chamber. For aerobic cultures, optical density at 660 nm was measured on a Jenway 7200 spectrophotometer (Bibby Scientific).
7
Extracellular Metabolite Quantification and Aerobic/Anaerobic Growth Measurement
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Extracellular metabolite concentrations were measured by high performance liquid chromatography as described by Verhoeven et al. [103 (link)] Optical density at 660 nm of aerobic cultures was measured using an Jenway 7200 spectrophotometer (Bibby Scientific, Staffordshire, UK) after accurate dilution to an OD660 between 0.1 and 0.3. Anaerobic cultures were first diluted to an optical density at 600 nm (OD600) between 0.15 and 0.35, followed by optical densities measurements at 600 nm on an Ultrospec 10 cell density meter (Biochrom, Harvard Biosience, Holliston, MA) that was placed in the anaerobic workstation [102 (link)].
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