In batch culture, all the medium is added at once without replenishment or replacement. With the active growth of microorganisms, the nutrients in the medium are gradually depleted, harmful metabolites continue to accumulate, and the logarithmic growth period of bacteria cannot be maintained for a long time. If fresh nutrients are constantly replenished in the incubator and the culture (including the bacterium and metabolites) is discharged at the same rate in a timely manner, the logarithmic growth period can theoretically be extended indefinitely.
Operation method
continuous culture method
Principle
In batch culture, all the medium is added at once without replenishment or replacement. With the active growth of microorganisms, the nutrients in the medium are gradually depleted, harmful metabolites continue to accumulate, and the logarithmic growth period of bacteria cannot be maintained for a long time. If fresh nutrients are constantly replenished in the incubator, and the culture (including the bacterium and metabolites) is discharged at the same rate in a timely and continuous manner, the logarithmic growth period can theoretically be extended indefinitely. As long as the outflow of culture solution can make the number of new bacteria increased by division and reproduction equal to the number of old bacteria outflow, it can ensure that the total bacterial population in the incubator is basically unchanged. The emergence of continuous cultivation methods can not only readily provide experimental materials of a certain physiological state for microbial research work, but also improve the production efficiency and automation level of the fermentation industry.
Materials and Instruments
Escherichia coli Move I. Main instruments, equipment and materials for the experiment For more product details, please visit Aladdin Scientific website.
Beef paste peptone medium Glucose NaCl Na2HPO4 (NH4)2SO4
Continuous Culture Unit
Simple continuous culture unit (see Figure 1)
Strain: Escherichia coli (E. Coli);
Medium: Basic medium: beef paste peptone medium;
Fermentation medium: glucose, NaCl, Na2HPO4, ( NH4)2SO4.
II. Experimental methodology, operational steps
1. Determination of reducing sugars: Colorimetric determination of 3,5-dinitrosalicylic acid (see experiments for specific procedures). Solid-state fermentation experiment of amylase) 2. Determination of protein concentration: Coomassie blue staining method
3. Determination of the proliferation curve of Escherichia coli (see the experiment for specific procedures) Determination of bacterial proliferation curve)
E. coli was inoculated in the parent medium, incubated at 37 ℃ for 6 h, the culture solution was centrifuged at 3 000 rpm for 10 min, the supernatant was decanted, and sterile saline was added to make a homogeneous solution with the cell number of 107 cells/ml, which was inoculated into the fermentation medium and incubated under the same conditions, and sampling was carried out at certain intervals, and the same culture solution with sterility was used as the control group. 600 nm wavelength colorimetry (required extinction between 0.3-0.6, if more than the appropriate dilution with the uninoculated culture medium), and then dry the culture medium in an infrared oven at 110-120 ℃ until constant weight, with the OD value of bacterial suspensions as the vertical coordinate and the corresponding concentration of the bacteria as the horizontal coordinate, plot a straight line, and find the slope 1/K.
K = biomass/OD
4. Pour the prepared fermentation medium into a 2 L triangular flask. Fix the glass tube with a cotton plug, and connect the top end of the glass tube to a rubber tube with an inner diameter of 3 mm and an outer diameter of 5 mm. Clamp with a spring clip and sterilize.
5. Sterilized triangular flasks cooled to 30 °C were mounted in a thermostatic water bath for incubation.
6. Connect the E. coli culture solution to the triangular flask with 10% inoculum, and carry out intermittent culture with stirring first, or stirring culture with aeration if there is a sterile air catheter.
7. Put 3 L of medium for replenishment in a 3 L triangular flask, insert one end of the rubber tube for feeding, fix it with a cotton plug, wrap the other end with oil paper and sterilize it.
8. Remove and measure the concentration of the intermittent culture at regular intervals to determine the rate of proliferation.
μ=dx/xdt
⇒ μt=dx/x ⇒ μ(t2-t1) = ln(x2/x1) ⇒ µ= (lnx2-ln x1)/ (t2-t1) 9. Remove the rubber tube for infusion, peel off the kraft paper paper, and peristaltic pump inlet connection, the incubator infusion rubber tube to the peristaltic pump outlet.
10. Depending on the rate of proliferation of the organisms, the medium is started at a smaller dilution rate, which must be less than this rate of proliferation.
F≤μ⇒F≤(lnx2-ln x1)/ (t2-t1)
III. Experimental results
1. Enter the measured OD600 values and the corresponding biomass in the table below.
2. Plot the time curves of reducing sugars, protein concentration and biomass during continuous fermentation of E. coli (indicate when to replenish).
