Modeling bacterial growth is important in maximizing the efficiencies of biological processes. Although there are many different methods of modeling bacterial growth, this experiment focuses on the Monod equations. However, in order to use the Monod equations, the maximum growth rate and Monod constant must be found. Here we show how the maximum growth rate and Monod constants can be obtained for Escherichia coli using M9 media in a bioreactor at 37 °C and 500 RPM. The maximum growth rate is also obtained at other settings to show how other factors can affect bacterial growth.
The maximum growth rate and Monod constants for E. coli with these settings were found to be – 0. 0600 hr-1 and – 4. 2 g/L respectively. With the Monod constant and maximum growth rate found, an E. coli population at 37°C and 500 RPM in M9 media can be modeled using the Monod equations. Introduction and Theory: Modeling bacterial growth can be a crucial step in maintaining and optimizing biological systems. Microorganisms can be modified to give a number of useful products. They are used in fermentation processes or can be genetically engineered to produce insulin, human growth hormone, or other important ormones and enzymes.
As with any process, it is desired to maximize the production. Knowing how to model and manipulate the bacterial growth will help to achieve this goal. There are many different ways to model bacterial growth, but this report will just focus on the Monod equation. The objective of this experiment is to find and compare the Monod constants and maximum growth rates for Escherichia coli under multiple conditions. This will allow the bacterial growth to be modeled. If this growth rate can be readily modeled, then the same methods should apply to genetically modified E. coli that roduce desirable products such as insulin.
The Monod equation1 can be written: dX/dt=p_g X=(H_max S)/(K_s+S) X where X is the biomass concentration, p_g is the specific growth rate, p_max is the maximum growth rate, S is the substrate eqn. 1 concentration and Ks is the Monod constant. The substrate concentration can be found at any time by knowing the yield coefficient, Y-¬¬¬¬X/s, the initial substrate concentration, SO, the initial biomass, X¬0 and the biomass at that time using the following equation1: S=(Y_(x/s) S_0-X+X_0)/Y_(x/s) eqn. 2 According to literature, the Yx/s in a glucose limited media for E. li can be estimated at 0. 5 grams biomass per gram glucose–2. The biomass will be estimated from values obtained from a spectrophotometer. When set at 600 nm, 1 OD will be approximately 0. 46 grams biomass per liter3. The relationship between optical density and bacteria population size is assumed to be linear. Bacterial population growth occurs over four stages as shown in Figure 1. The first stage is the lag phase. This is where the bacteria adjust to the new media before beginning growth. Different genes will activate to allow the new media to be metabolized.
The second stage is the exponential phase. This is where the cells begin to metabolize the media and cell growth is most rapid. Most of the experimental data was collected during this phase. In the third stage, the stationary phase, bacterial growth halts. The cells run out of nutrients and cannot continue to grow. Finally, there is an accumulation of toxins which begin to kill the cells. This fourth stage is called the death phase. Figure 1. A basic example of how biomass concentration changes over time with the different stages of bacterial population growth labeled.
Different variables were altered to determine their effect on the acterial growth rate. The main variables to be studied were stir rate, temperature, method of growth, and media. The stir rate is important because it will affect how much oxygen is delivered to the cells and will also help distribute nutrients equally. The temperature is important because a higher temperature is associated with faster cell growth. However, too high of a temperature will denature the proteins in the cell and kill the bacterial population. Another variable was the media choice.
The two media used in this experiment were M9 and LB. The M9 media is a defined media. This means its contents are known. The M9 media can be manipulated to have a certain glucose concentration which makes it easier to study the substrate concentration. Contrary to the defined media, LB is a complex media. Complex media often contain biological components such as beef extracts or blood4. This means that the exact contents of the LB media are unknown and as such the substrate concentration is also unknown. Finally, there were different methods of growing the bacteria culture.
One method was the benchtop shaker method. This consisted of multiple flasks stirring in an incubator unit that ontrolled temperature and rotating speed. The multiple flasks allowed for more data collection, which will prove the experiment is repeatable. The other method of growing bacterial cultures was to use a bioreactor. The bioreactor is capable of controlling and measuring many different variables such as temperature, stir rate, and pH with the right attachments. Although pH and dissolved oxygen concentration play an important part in cell growth, these were not varied for this experiment.
The differences between the bioreactor and benchtop shaker methods will be explained more in the Apparatus and Procedure section. Apparatus and Procedure: Figure 2. Equipment used in experiment. A) Autoclave – If used correctly, this will sterilize the equipment and prevent contamination. B) Biosafety hood – Inoculations should be done here as well as measurements for the benchtop shaker method to prevent contamination. C) Bioreactor – stirs as well as uses a temperature probe and heating/cooling waters to change the temperature. D) Incubator and Shaker Unit – Shakes and heats the flasks to the desired settings.
The equipment needed for this experiment includes a bioreactor unit, a benchtop shaker unit, a pectrophotometer, flasks, graduated cylinders, cuvettes, micropipettes and tips, aluminum foil, and autoclave tape. Figure 2 displays some of these. Before the experiment began, the lab equipment was cleaned and sterilized. The bioreactor was disassembled and all equipment was rinsed thoroughly. Then, aluminum foil and autoclave tape was used to cover all openings in the equipment. After all the equipment had been cleaned and all the holes were covered, the equipment was autoclaved. Next, the cell cultures must be grown overnight.
This portion should take place in the biosafety hood. All necessary equipment was properly sterilized for inoculation before placing them inside the biosafety hood. The E. coli solution was warmed until it was completely melted. Then, 60 mL of media was added to each flask along with 1 mL of the E. coli culture. The flasks were covered with the aluminum foil and placed in the incubator. The temperature was set at 37 °C and the mixing speed was set at 200 RPM to grow overnight. After the cultures had grown for around 15 hours, the bioreactor and benchtop shakers were started.
The bioreactor was reassembled and the cooling water was primed. 0 mL of media was added to the bioreactor using sterilized equipment. After the reactor reached the desired temperature, 50 mL of the inoculated bacteria was added. For the benchtop shaker method, 95 mL of media was each flask along with 5 mL of inoculated bacteria. Using pure media as a blank, samples were taken every 30 minutes at an absorbance reading of 600 nm. After the absorbance was read, the sample was dumped into the biowaste container. Once the readings have been stable for around one hour, the culture was assumed to be in the stationary phase and data collection ended.
Finally, the steps at he beginning were repeated to properly clean and sterilize the equipment. Safety Considerations: The bioreactor and benchtop shaker both have moving parts that should be monitored. However, if used correctly, the moving parts will not be an issue. Both also use electricity, which could cause problems if there is improper wiring. Also, an autoclave is used for sterilization. Because the autoclave needs high pressures and temperatures to kill the bacteria, it must be used properly to avoid injury. Ethanol, bleach, and Alconox are used for cleaning and could have negative health effects if used mproperly.
Ethanol can cause skin or eye irritation and ingestion may cause nausea, vomiting, diarrhea and may affect the brain, liver, blood, and heart. Bleach can cause serious eye damage or irritation and can cause skin corrosion and irritation. Alconox can cause skin or respiratory irritation. In case of accident, rinse the affected areas for at least 15 minutes with water. Safety goggles, a lab coat, and gloves are required for this experiment to reduce the chances of spilling chemicals on the skin, eyes, or other important body parts. They will also help prevent contamination.
