Shake-flask cultures were performed in 500 mL shake flasks containing 100 mL of SM with 20 g L−1 sucrose, in an Innova incubator shaker (New Brunswick Scientific, Edison, NJ, USA) set at 200 rpm and at 30°C under an air atmosphere. For growth rate determinations, cells were inoculated in SM with 20 g L−1 glucose from a frozen stock culture. After reaching stationary phase, the culture was transferred to SM with 20 g L−1 sucrose (initial OD660nm = 0.2) and incubated until exponential growth was observed. Exponentially growing cultures were then transferred to fresh medium (initial OD660nm = 0.2) and samples were taken hourly until stationary phase was reached. Optical density at 660 nm was measured with a Libra S11 spectrophotometer (Biochrom, Cambridge, UK). Specific growth rates were calculated from at least five data points.
Libra s11 spectrophotometer
Manufactured by Harvard Bioscience
Sourced in United Kingdom
The Libra S11 spectrophotometer is a compact and efficient instrument designed for accurate absorbance measurements. It provides a reliable means of analyzing the concentration and purity of various samples, including proteins, nucleic acids, and other biomolecules. The Libra S11 spectrophotometer offers a wavelength range of 190-1100 nm, allowing for a wide variety of applications in the life sciences and analytical chemistry fields.
Automatically generated - may contain errors
Lab products found in correlation
Innova incubator shaker, by Eppendorf (2 mentions)
Pluronic pe 6100 antifoam, by BASF (1 mentions)
Type a e, by Pall Corporation (1 mentions)
Microwave oven, by Bosch (1 mentions)
L carnitine, by Merck Group (1 mentions)
Fungalight am cfda, by Thermo Fisher Scientific (1 mentions)
Cell lab quanta sc mpl flow cytometer, by Beckman Coulter (1 mentions)
Pageruler prestained protein ladder, by Thermo Fisher Scientific (1 mentions)
Dionex ultimate 3000 system, by Thermo Fisher Scientific (1 mentions)
Syncronis c18 column, by Thermo Fisher Scientific (1 mentions)
Luna c8 2 column, by Phenomenex (1 mentions)
12 protocols using libra s11 spectrophotometer
1
Shake-Flask Cultivation of Microorganisms
2
Bioreactor Cultivation Analysis Methods
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Off-gas analysis, biomass dry weight measurements, HPLC analysis of culture supernatants and correction for ethanol evaporation in bioreactor experiments were performed as described previously [20 (link)]. Optical density was determined at 660 nm, using a Libra S11 spectrophotometer (Biochrom, Cambridge, United Kingdom). In batch cultures, yields of products were calculated from samples taken at mid-exponential phase (minimum of five samples), as described previously [39 (link)]. Biomass and product yields in chemostat cultures were determined from residual glucose, biomass and metabolite concentrations in steady-state cultures, analysed after rapid quenching of culture samples [40 (link)].
For calculation of degree of reduction (electron) balances in cultures, the degrees of reduction of biomass, CO2, NH4+ and extracellular metabolites (glucose, ethanol, glycerol, succinate, pyruvate, lactate and acetate) were defined as described in [41 (link)].
Estimations of statistical significance of differences in yields between strains were determined with two-tailed Student’s t tests. All values are represented as averages ± mean deviation of independent biological replicate cultures, performed at least in duplicate.
For calculation of degree of reduction (electron) balances in cultures, the degrees of reduction of biomass, CO2, NH4+ and extracellular metabolites (glucose, ethanol, glycerol, succinate, pyruvate, lactate and acetate) were defined as described in [41 (link)].
Estimations of statistical significance of differences in yields between strains were determined with two-tailed Student’s t tests. All values are represented as averages ± mean deviation of independent biological replicate cultures, performed at least in duplicate.
3
Shake-flask and Chemostat Cultivation of Yeast
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Shake-flask cultures were grown at 30°C in 500-ml flasks containing 100 ml synthetic medium (47 (link)) with 20 g liter−1 glucose in an Innova incubator shaker (New Brunswick Scientific, Edison, NJ) set at 200 rpm. When required, media were supplemented with lipoic acid at the concentration of 50 ng ml−1. Optical density at 660 nm was measured at regular time intervals with a Libra S11 spectrophotometer (Biochrom, Cambrige, United Kingdom). Chemostat cultivations were carried out at 30°C in 2-liter laboratory bioreactors (Applikon, Schiedam, The Netherlands) with working volumes of 1 liter. Chemostat cultivation was preceded by a batch phase under the same conditions. When a rapid decrease in CO2 production indicated glucose depletion in the batch cultures, continuous cultivation at a dilution rate of 0.10 h−1 was initiated. Synthetic medium (47 (link)) supplemented with 7.5 g liter−1 glucose was used. Lipoic acid solution in ethanol was prepared separately and added to the medium to a final concentration of 500 ng ml−1. Antifoam Pluronic PE 6100 (BASF, Ludwigshafen, Germany) was added to the media before sterilization to a final concentration of 0.15 g liter−1. Culture pH was maintained at 5.0 by automatic addition of 2 M KOH. Aerobic bioreactors were sparged with 500 ml min−1 air and stirred at 800 rpm to ensure fully aerobic conditions.
4
Biomass Determination for Fungal and Yeast Cultures
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Penicillium chrysogenum biomass dry weight was determined in duplicate by filtration of 10 mL culture sample over pre-weighed glass fibre filters (Type A/E, Pall Life Sciences, Hoegaarden, Belgium). After filtration, filters were washed with demineralized water and dried for 10 min at 600 W in a microwave oven (Bosch, Stuttgart, Germany) prior to reweighing. Biomass dry weight in S. cerevisiae culture samples was determined with a similar procedure using nitrocellulose filters (0.45-µm pore size; Gelman Laboratory, Ann Arbor, MI) and drying for 20 min in a microwave oven at 360 W output. Optical density (OD) of the cultures was determined at 660 nm with a Libra S11 spectrophotometer (Biochrom, Cambridge, United Kingdom). Determination of CO2 and O2 concentrations in the bioreactor exhaust gas and HPLC analysis of metabolite concentrations in culture supernatant samples were performed as described previously [50 (link)].
5
Growth Kinetics of Microbial Strains
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Growth studies were conducted at 30°C in 500-mL flasks. To prevent nutrient carry over from stock cultures, strains were pre-grown in two sequential shake flasks in which biomass formation was not limited by the amount of carbon source but by the nitrogen source. To this end, 200-μL cell suspension from frozen stocks were inoculated in 100 mL SM-glutamate with a decreased initial l -glutamate concentration of 1.5 mM and with 20 g⋅L−1 glucose. After reaching stationary phase, biomass was centrifuged (4°C, 5 min at 3000 g). The pellet was washed twice with demineralized water, resuspended in demineralized water and used to inoculate a second shake flask with the same medium. After reaching stationary phase, the same wash procedure was performed and a final set of shake flasks, containing either 100 mL SM-urea or SM-glutamate and 20 g⋅L−1 glucose, was inoculated for growth rate determination,. Where indicated, l -carnitine (Sigma-Aldrich) was added at a final concentration of 0.4 g⋅L−1. Optical density at 660 nm was measured at regular time intervals with a Libra S11 spectrophotometer (Biochrom, Cambrige, UK).
6
Biomass Growth Monitoring in Chemostat and Fed-Batch Cultures
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For the chemostat cultures, biomass growth was monitored by optical density (OD) measurement at a wavelength of 660 nm with a Libra S11 spectrophotometer (Biochrom). For the fed‐batch cultures, biomass growth was monitored by OD measurement at a wavelength of 600 nm with a Thermo Genesys spectrophotometer (Thermo Fisher Scientific).
For the chemostat cultures, dry weight was determined by filtering 10 ml culture broth over a preweighed nitrocellulose filter with a pore size 0.45 μm, washing the filter with demineralized water and drying the filter for 20 min at 360 W in a microwave oven before weighing again (Postma et al., 1989 (link)). Duplicate measurements varied less than 3.5% throughout the cultivation. For the fed‐batch cultures, dry weight was determined by centrifuging 2 × 5 ml of culture broth at 6,000g for 15 min. The pellet was washed once by resuspending in deionized water and centrifuged again at 6,000g for 15 min. After washing, the pellet was dried for 24 h at 105°C and weighed.
For the chemostat cultures, dry weight was determined by filtering 10 ml culture broth over a preweighed nitrocellulose filter with a pore size 0.45 μm, washing the filter with demineralized water and drying the filter for 20 min at 360 W in a microwave oven before weighing again (Postma et al., 1989 (link)). Duplicate measurements varied less than 3.5% throughout the cultivation. For the fed‐batch cultures, dry weight was determined by centrifuging 2 × 5 ml of culture broth at 6,000g for 15 min. The pellet was washed once by resuspending in deionized water and centrifuged again at 6,000g for 15 min. After washing, the pellet was dried for 24 h at 105°C and weighed.
7
Bacterial Growth on Glucose and Arabinose
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Thawed 1-mL aliquots from frozen stock cultures were used to inoculate shake flask precultures on SM-urea supplemented with either d -glucose (20 g L−1), l -arabinose (20 g L−1), or both sugars (both 20 g L−1). These precultures were used to inoculate a second culture which was subsequently used to inoculate a third culture which was inoculated at an initial OD660 of 0.1 and used to monitor growth. Optical densities at 660 nm were measured with a Libra S11 spectrophotometer (Biochrom, Cambridge, United Kingdom). Maximum specific growth rates (μmax) were derived from at least four consecutive data points derived from samples taken during the exponential growth phase of each culture.
8
Growth Studies: Glucose, Lipoic Acid, L-Carnitine
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For growth studies in shake flasks and using spot plates, strains were pregrown in shake flasks with SM-urea and 20 g ⋅ liter−1 glucose with lipoic acid or l -carnitine, where appropriate. For growth studies in shake flasks, cells were washed twice with synthetic medium (61 (link)) and transferred to new shake flasks with SM-urea containing 20 g ⋅ liter−1 glucose and 40 mg ⋅ liter−1l -carnitine or 50 ng ⋅ liter−1 lipoic acid, where indicated. Growth rates were based on optical density at 660 nm (OD660) measurements using a Libra S11 spectrophotometer (Biochrom, Cambridge, United Kingdom). Culture viability was estimated with the FungaLight AM-CFDA (acetoxymethyl ester 5-carboxyfluorescein diacetate)/propidium iodide yeast viability kit (Invitrogen, Carlsbad, CA) and a Cell Lab Quanta SC MPL flow cytometer (Beckman Coulter, Woerden, The Netherlands) as described previously (73 (link)). For the preparation of spot plates, precultures were washed once with synthetic medium and diluted in synthetic medium to an OD660 of 0.273 (corresponding to 2 × 106 cells ⋅ ml−1). Five-microliter samples of a dilution series, containing an estimated 2 × 105, 2 × 104, and 2 × 103 cells per ml, were spotted on SM-urea agar plates with 20 g ⋅ liter−1 glucose and l -carnitine (400 mg ⋅ liter−1) or lipoic acid (50 ng ⋅ liter−1) as indicated.
9
Quantification of Biomass, Protein, and Steroids
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Suspended biomass concentrations were determined photometrically by measuring the optical density at a wavelength of 450 nm (Libra S11 spectrophotometer, Biochrom Ltd., Cambridge, UK). One OD450 unit corresponds to 0.166 gCDW L−1 (Blank et al., 2008 (link)).
Monitoring of protein synthesis was carried out by harvesting 80 μg of cell dry weight (CDW) from cultures for sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) according to Laemmli (Laemmli, 1970 (link)). Proteins extracted from 15 μgCDW were loaded per lane and stained with Coomassie Brillant Blue R‐250. PageRuler™ Prestained Protein Ladder (Thermo Fisher Scientific) was used as reference.
Glucose availability in resting‐cell reaction mixtures was checked with Medi‐Test glucose test stripes (Macherey‐Nagel).
Steroid concentrations were determined via HPLC using a Dionex Ultimate 3000 system (Thermo Fisher Scientific) equipped with a Syncronis C18 column (150 x 2.1 mm, 3 μm particle size, Thermo Fisher Scientific) and an UV detector operating at 245 nm for steroid detection. Steroids (5 μL injection volume) were quantified by elution at a column oven temperature of 40°C with 55% acetonitrile in ultrapure water as a mobile phase at a flow rate of 0.5 mL min−1. Steroid concentrations were calculated based on peak areas and calibration curves established with commercially available standards.
Monitoring of protein synthesis was carried out by harvesting 80 μg of cell dry weight (CDW) from cultures for sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) according to Laemmli (Laemmli, 1970 (link)). Proteins extracted from 15 μgCDW were loaded per lane and stained with Coomassie Brillant Blue R‐250. PageRuler™ Prestained Protein Ladder (Thermo Fisher Scientific) was used as reference.
Glucose availability in resting‐cell reaction mixtures was checked with Medi‐Test glucose test stripes (Macherey‐Nagel).
Steroid concentrations were determined via HPLC using a Dionex Ultimate 3000 system (Thermo Fisher Scientific) equipped with a Syncronis C18 column (150 x 2.1 mm, 3 μm particle size, Thermo Fisher Scientific) and an UV detector operating at 245 nm for steroid detection. Steroids (5 μL injection volume) were quantified by elution at a column oven temperature of 40°C with 55% acetonitrile in ultrapure water as a mobile phase at a flow rate of 0.5 mL min−1. Steroid concentrations were calculated based on peak areas and calibration curves established with commercially available standards.
10
Bioreactor Cultivation and Analytics
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Determination of optical density at 660 nm was done using a Libra S11 spectrophotometer (Biochrom, Cambridge, UK). Off-gas analysis, biomass dry weight measurements, HPLC analysis of culture supernatants and correction for ethanol evaporation in bioreactor experiments were performed as described previously [20 (link)]. For anaerobic batch cultures, biomass concentrations were estimated from OD660 measurements, using calibration curves based of a minimum of six samples taken in mid-exponential phase for which both biomass dry weight and OD660 were measured. Yields of each fermentation were calculated from a minimum of six samples taken during the mid-exponential growth phase by plotting either biomass against substrate, ethanol against substrate, glycerol against substrate, acetate against substrate, glycerol against biomass or acetate against biomass and calculating the absolute value of the slopes of the resulting linear fits. An example of the calculations performed is given in Additional file 2 : Table S2.
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