Home Virus Germs & Bacteria Analysis of the influence of flame sterilization included in sampling operations on shake-flask cultures of microorganisms

Analysis of the influence of flame sterilization included in sampling operations on shake-flask cultures of microorganisms

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Analysis of manual flame sterilization in sampling process using CDMSS

Manual flame sterilization was performed using a Bunsen burner, 500 mL Erlenmeyer flask with small pipes on the sides, and CDMSS. During the flame sterilization process, the gas in the headspace of the flask was monitored using CDMSS by circulation at 50 mL/min, and the maximum CO2 concentration was recorded. The manual flame sterilization was performed for 5, 10, and 20 s; each time point was repeated four or more times, and the average value was calculated. All experiments were performed at room temperature (approximately 25 °C).

Analysis of flame sterilization operation with fixed operating angle and time using CDMSS

The system shown in Supplementary Fig. S1 online was constructed, and the effect of flame sterilization on the CO2 concentration in the headspace of the flask was analyzed. Although the flask was brought near the Bunsen burner in the manual experiments, in this system, the position and angle of the flask were fixed when the Bunsen burner was brought close to the flask. After the operation time, the slide plate was moved to separate the Bunsen burner from the flask while monitoring the headspace of the flask using CDMSS (see Supplementary Fig. S1 online, Figs. 1–6), and the maximum CO2 concentration was recorded. The operation time and inclination angle were set with reference to the manual flame sterilization procedure, namely 5, 10, or 20 s, and 15, 25, or 35°, respectively. All experiments were performed at room temperature in four replicates, and the average values were calculated.

Measurement of CO2 concentration generated by flame sterilization procedure mimicked using CDMSS and AAFS

In order to investigate the effect of only the flame sterilization operation on the shake-flask culture without the other sampling processes, we connected a 99.8% (v/v) CO2 gas cylinder to the AAFS to aerate intermittently the headspace of the shaking flask15. The CO2 concentration in the gas phase of the flask was monitored using CDMSS under various intermittent aeration conditions, and the maximum CO2 concentration was recorded. All experiments were performed at room temperature in four replicates, and the average values were calculated.

Computational domain and settings

Supplementary Fig. S6 online shows the schematics of the computational domain. A Bunsen burner, with a pipe size of 12.7 mm for its outer diameter and a thickness of 1.00 mm, was installed upwards. The capacity of the Erlenmeyer flask was 500 mL, and it was empty. The lower part of the flask tip was installed 60 mm above the burner rim top and the flask was tilted 60 °. The X, Y, and Z directional sizes of the computational domain were 450 mm, 320 mm, and 370 mm, respectively, and the domain was divided into 920,000 cells.

Calculations were conducted by using Fluent 2019R1 (ANSYS Inc., Canonsburg, U.S.A.). The physical modeling was as follows. The transport equations of mass, momentum, energy, and species were solved as laminar flow, and CH4, O2, CO, CO2, H2O, and N2 were considered chemical species, with their thermal properties depending on temperature. Westbrook and Dryer’s two-step reaction mechanism was employed32. Gravity was also considered. The time step was 5 × 10−4 s and the maximum iteration number per time step was 20. The calculation was continued until the flow time reached 20 s.

As boundary conditions, the inlet flow rate of the Bunsen burner was 6.53 × 10−5 m3/s at standard state. The fuel was CH4 and the oxidizer was air, which consisted of 21.0% O2 and 79.0% N2. The equivalence ratio Φ of the premixed gas was 2.00. Here, the equivalence ratio is defined as (varPhi =frac{frac{{X}_{CH4}}{{X}_{O2}}}{{left(frac{{X}_{CH4}}{{X}_{O2}}right)}_{st}}), where Xi is the mole fraction of species i, the subscript st denotes stoichiometric conditions, and ({(frac{{X}_{CH4}}{{X}_{O2}})}_{st}) is 0.5. For the remaining lower interface, 0.100 m/s was set as the upward ambient co-flow; the component was air. The temperature of these gases was 26.85 °C. The surrounding boundaries other than the lower parts were set as pressure outlets, and when inflowing from outside, the gases were air at 26.85 °C. The flask and burner were made of borosilicate glass and 304 stainless steel, respectively. For the borosilicate glass, the density, heat capacity, and thermal conductivity were 2,190 kg/m3, 740 J/kg-K, and 1.38 W/m-K, respectively. The corresponding values for the stainless steel were 7,930 kg/m3, 590 J/kg-K, and 16.7 W/m-K, respectively.

In the simulation process, a premixed flame was first formed without installation of the flask. After the flask was positioned, the temperature of the flask and inside the flask was set as ambient temperature and the inside filled with air. This time was defined as t = 0 s and a transient calculation was conducted.

Empirical verification

A simple experiment was also conducted for verifying the accuracy of the simulation. The sizes and positions of the burner and flask were the same as those in the simulation in the previous section. The fuel was CH4 with a purity of > 99.5% and the oxidizer was ambient air. The flow rate of the fuel was controlled by mass flow meter, which was 0.680 L/min at standard state.

Supplementary Fig. S7 online compares the flame shapes between CH4 distributions in the simulation and a direct photograph of the experiment, without the flask. The flame shape in the simulation qualitatively corresponds with that in the experiment. Two flames, an inner and an outer flame, are formed. The height of the inner flame is approximately 40 mm. For the outer flame, although the height cannot be accurately measured because its tip is unclear, it is presumed to be approximately 80 mm. Supplementary Fig. S8 online shows the experimental setup for heating the flask. The flask was suspended by thin wires. Four K-type sheathed thermocouples (1.0 mm in diameter) were installed inside the flask. In order to measure the surface temperature of the flask using a non-contact infrared thermography device, the flask was colored black. Supplementary Fig. S9 online shows the temperature along the center axis of the flask at t = 20 s. In the experiment, these temperatures were measured at heights of 42, 72, 102, and 142 mm (see Supplementary Fig. S9 online, closed red circle). The temperatures for both the simulation and experiment were ambient from 0–100 mm. At > 120 mm, the temperature drastically increased in the simulation, approaching 1,400 °C at 172 mm. In contrast, in the experimental result, the temperature at 142 mm was 75.2 °C, which was 130 °C lower than the corresponding simulation. There are two main reasons that the temperature in the simulation was higher. First, a two-step reaction mechanism was used. For Φ = 2.0, the adiabatic flame temperature was 270 °C higher than the value when employing detailed mechanisms. Second, the dilution of burned gas by co-flows was different from that occurring in a real situation. Here, 0.1 m/s was set as the co-flow velocity because a calm flow field was assumed; flow fields might be more turbulent in the laboratory. In addition, according to the infrared thermography device, the maximum temperature of the flask exceeded 500 °C at t = 180 s, but the area where the temperature was > 100 °C was limited to the neck regions with heights > 130 mm.

As indicated above, we confirmed that our simulation can qualitatively evaluate the behavior of burned gas around the flask.

Microorganisms and medium

E. coli K12 IFO3301, P. saccharophila NBRC103037, A. pasteurianus NBRC3283, and S. cerevisiae IFO0309 were used in this study. The LB medium used for E. coli cultivation consisted of (g/L): tryptone, 10; yeast extract, 5; and NaCl, 5. The R2A medium used for P. saccharophila cultivation consisted of (g/L): yeast extract, 0.5; peptone, 0.5; casamino acids, 0.5; glucose, 0.5; soluble starch, 0.5; sodium pyruvate, 0.3; K2HPO4, 0.3; and MgSO4·7H2O, 0.05. The NBRC no. 804 medium used for A. pasteurianus cultivation consisted of (g/L): hipolypepton, 5; yeast extract, 5; D-glucose, 5; MgSO4·7H2O, 1. The YM medium used for S. cerevisiae cultivation consisted of (g/L): D-glucose, 10; peptone, 5; yeast extract, 3; and malt extract, 3.

Inoculum preparation

A loop-full of E. coli IFO3301, P. saccharophila NBRC103037, A. pasteurianus NBRC3283 or S. cerevisiae IFO0309 slant culture was inoculated into a 500 mL Erlenmeyer flask containing 100 mL of LB, R2A, NBRC no. 804 or YM media, respectively. The samples were then cultivated at 30 °C on a rotary shaker with a 70-mm shaking diameter at 100 rpm for 6.75 h (E. coli) or 20 h (P. saccharophila) and 200 rpm for 72 h (A. pasteurianus) or 14 h (S. cerevisiae). Glycerol stocks were prepared by adding the culture medium to glycerol (final concentration 20% [v/v]) and stored at −80 °C.

Culture conditions

One millilitre of each glycerol stock was inoculated into a 500 mL Erlenmeyer flask containing 100 mL of 4 media types and cultured at 30 °C on a rotary shaker with a 70-mm shaking diameter at 100 rpm (E. coli and P. saccharophila) or 200 rpm (A. pasteurianus and S. cerevisiae). For air permeability, the Erlenmeyer flask was equipped with a BIO-SILICO N-38 sponge plug (Shin-Etsu Polymer Co., Ltd, Tokyo, Japan; breathable culture-plug). After each sampling of the culture broth, 99.8% (v/v) CO2 gas was aseptically aerated into each flask headspace using the AAFS for either 90 s or 30 s at 30 mL/min.

Sampling of culture broth during shake-flask culture

Conventional sampling methods, which include interruption of shaking, opening the culture-plug, and flame sterilization on a clean bench, are known to affect the cultured microorganisms. Therefore, sampling during the shake-flask culturing was performed with the system shown in Supplementary Fig. S10 online, which is similar to the CDMSS sampling method5. Specifically, the sampling unit and flask were autoclaved independently. Then, the sampling unit, flask, three-way plug, and 50 mL syringe with a 0.2 μm filter were assembled on a clean bench, as shown in Supplementary Fig. S10 online. The three-way plug, 50 mL syringe, and 0.2 μm filter were disposable. Sampling of the shake-flask culture was performed by attaching a syringe to a three-way plug connected to a sampling tube. After collecting the culture broth, in order to ensure its removal from the sampling tube, a volume of fresh air (0.2 μm filtered) was used to flush the broth out of the tube. The sampled culture broth was stored at −80 °C until used for analysis.

Measurement of culture factors

The U.O.D.600 and pH of the culture broth, which was sampled without interruption of shaking, were measured using a V-570 spectrophotometer (JASCO, Tokyo, Japan) and a pH meter (HORIBA, Kyoto, Japan), respectively. In order to minimize the decrease in volume of the culture broth due to repeated sampling, the total sampled volume did not exceed 10% (v/v) of the total volume of the initial culture medium. All measurements were performed in duplicate. In the case of P. saccharophila, the culture broth contained cell aggregates; these aggregates were disrupted by vortex and ultrasonication for 10 min at 4 °C prior to absorbance measurements.

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