Shake Flask Fermentation

Table of Contents

1. Abstract
2. Introduction
3. Aims
4. Theory
5. Apparatus
6. Procedure
7. Result
8. Calculation
9. Discussion
10. Conclusion
11. Recommendation
12. References

Abstract

The growth kinetic of microorganism in shake flask is studied using Escherichia coli as a sample. Microorganism in different volumes is transferred into 250ml Erlenmeyer/shake flasks containing nutrient media. The effect on the constant volume of media used is determined by the varying volume of microorganism transferred.

There are three methods for testing the growth kinetic rate of microorganisms in shake flasks: optical density (OD) measurement, glucose analysis, and cell dry weight (CDW) determination. These tests provide data for plotting the growth curve graph, which is then used to compare and analyze the growth curve of the microorganism.

Introduction

Shake flask fermentation is a widely used fermentation method for screening high-producing strains in laboratory settings. It involves using a small volume of nutrient broth to conduct the fermentation.

Lysogeny broth or luria bertani (LB) is a nutrient-rich medium commonly utilized for bacterial growth. Typically, a shake flask with a volume ranging from 250ml to 500ml is employed. Previous research has revealed that smaller flask volumes result in improved oxygen transfer rates, but this is only suitable for short-term fermentation as the medium may evaporate. Various designs of shake flasks are available, including the standard Erlenmeyer flask, flying saucer shake flask, shake flask with baffles, and flat bed Thompson bottle. For this particular experiment, the standard shake flask design is chosen.

Erlenmeyer flask is equipped with cotton wool stoppers and autoclaved along with the nutrient broth. The flask is then cooled to room temperature before the microbes are added and placed into the shaker machine. Many shaker machines are compatible with flask fermentation, while some can be adjusted for other uses. Another important component in shake flasks is plugs, which come in various types such as cotton-wool, glass wool, polyurethane foam, or synthetic fibrous material.

The purpose of plugs is to protect the medium from airborne microorganisms while allowing air to flow into the flask. A shaker machine is utilized to aid in oxygen transfer for aerobic respiration microorganisms. For a more precise environment, an incubator shaking cabinet is ideal as it can regulate temperature, lighting, gas levels, and humidity. The shaker’s movement is typically rotary or reciprocating, and increasing its speed enhances the oxygen transfer rate in a flask.

Aims

The objective is to study the growth kinetics of microorganisms in a shake flask experiment. This includes constructing a growth curve that incorporates the different phases of growth, namely the lag phase, log phase, stationary phase, and death phase. Additionally, the aim is to determine the Monod parameters.

Theory

Several techniques are necessary for growth culture in a shake flask to ensure safety and precision of the experiment. The first step involves preparing culture media, which should be done under a laminar flow safety cabinet. The transfer of culture to and from the media and cuvette must follow the aseptic technique to avoid introducing unwanted microorganisms into the media. To create a growth curve, the sample’s optical density is measured using a spectrophotometer, which is quick and easy. For samples with E.coli, it is crucial to set the wavelength at 600nm and record every reading. The growth curve can be represented by a diagram consisting of four distinct phases: lag phase, exponential phase, stationary phase, and death phase. The initial stage is called the lag phase characterized by slow growth where cell number remains constant while enzymes are activated to utilize substrate.

In order to enhance productivity, it is crucial to reduce the lag period in industry. Various techniques have been employed to shorten this lag phase, including inoculating with cells in the exponential phase, pre-acclimating the inoculum in growth media, and utilizing a large cell inoculum size. The next stage is the exponential phase where cells have adjusted to their surroundings and begin multiplying quickly. Furthermore, there is an ample supply of nutrients and substrates during this phase. The growth rate during this stage remains unaffected by the concentration of nutrients and substrates.

The exponential phase is characterized by exponential increases in cell number and mass concentration. It is situated between the log phase and the stationary phase, which is a deceleration phase caused by the depletion of one or more nutrients. The deceleration may be caused by the accumulation of toxic byproducts of growth. The growth and metabolism during this phase becomes unbalanced due to shifts for survival. The third phase, known as the stationary phase, is characterized by an equal rate of development of new cells and death, resulting in no net growth of cell number or mass. This phase does not involve cell division. Additionally, this stage is known for its production of secondary metabolites at a relatively high level.

Endogenous energy metabolism is capable of preserving the viability of cells, and the presence of extra substrate can eliminate the inhibitory compound. The death phase represents the stage where the rate of cell death exceeds the rate of new cell formation. During this phase, cell lysis occurs and growth can be restored by transferring to a fresh medium.

Apparatus

A microbe grown on an agar plate (E. coli) is placed in shake flasks. A sterile loop of wire is used for handling. The flasks are sealed with a cotton plug, which can be made of cotton, cotton mesh, or aluminium foil. The flasks are then placed in an incubator shaker. The media used for growing the inoculums is LB. A pippettor is used for transferring the media. Centrifuge tubes and cuvettes are used for various purposes. Falcon tubes are also utilized. Light from a Bunsen burner is used to sterilize items. Ethanol and distilled water are required for various sterilization procedures. A sterilized graduated cylinder is also necessary.

Procedure

Media preparation:

1. Luria Bertani (LB) broth (Lennox) is calculated to get concentration for 200ml of broth. LB brpth composition written to make media on the box that stored the LB is 10 g/L. So, 1. 6g of LB broth powder is diluted with 160ml of distilled water to make the media.
2. 160ml of the LB broth is prepared inside 250ml Erlenmeyer flask.
3. Erlenmeyer flask is closed with cotton covered by gauge and aluminum foil.
4. 10 g/L of glucose is diluted with the same amount of distilled water used to dilute media that is 160 ml and is placed in a schott bottle. ) The Erlenmeyer flask containing media with all the experiment is autoclaved for 3 hours.

Sampling for absorbance analysis or optical density:

1. 2 ml of inoculums is taken out and being transferred into cuvette.
2. 2 ml of blank (LB broth not contain microorganism) is transferred into cuvette.
3. The spectrophotometer is calibrated to zero by blank consisting of 2 ml pure LB broth (not contain microorganism).
4. Then optical density measurement of the inoculums is measured by setting the wavelength at the range of 600nm.
5. More absorbance means higher number of cell detected.

The sampling is repeated 12 times within a 24-hour period, with samples taken every 2 hours.

Analysis of glucose:

1. 2 ml of inoculums is transferred into micro centrifuge tube.
2. Sample is then centrifuge at 10000 rpm with temperature 4 0C for 10 minutes.
3. After centrifuge, cell (solid) will deposited at the bottom of the centrifuge tube and supernatant (liquid) is separated from the cell.
4. This supernatant is transfer into another centrifuge tube for glucose analysis; the cell will then followed the next test which is cell dry weight.
5. Centrifuge tube containing supernatant is then slotted in the slot of the YTI turntable Biochemical Analyzer in order to analyzer the glucose existed inside the supernatant.
6. After 24 hours, the inoculums is transferred back into 135 ml medium prepared making a total of 150 ml of inoculums prepared thus finishing the steps of preparation of inoculums.

Sampling for measuring the dry weight of cells:

1. All 12 centrifuge tube containing cell (solid) from procedure for glucose analysis is arranged on the centrifuge tube rack.
2. The cap is opened inserted into the oven.
3. Centrifuge tube rack containing centrifuge is put inside the oven at 60 0C for 24 hours.
4. The dried biomass is then being placed inside desiccators to let it cool before rapidly weighing on an analytical balance.

Before running the experiment, we must prepare for it in three stages: sterilization preparation, media preparation, and inoculum preparation. There are three methods used to identify the growth of bacteria or microorganisms: optical density, cell dry weight, and glucose analysis. The optical density test, or absorbance analysis, is performed using a spectrophotometer set at a wavelength of 600nm. A 2 ml sample is diluted and transferred to a cuvette to obtain the optical density reading.

The optical density (OD) reading is tabulated and the graph of OD is plotted in figure 2.0. The graph demonstrates a rapid increase in reading within the initial two hours, followed by a stable reading until the 22nd hour. Over the last 2 hours, there is a decrease in the reading. By comparing the results of the OD test to the microorganism growth curve, we can conclude that the first 2 hours correspond to the log phase or exponential phase, during which cell division occurs rapidly through binary fission. Additionally, during this phase, cells also begin to proliferate at a constant rate towards their maximum growth rate.

According to figure 1.0, the doubling time for E. coli is found to be 20 minutes in the growth phase, which signifies the most consistent and ideal growth. The lag phase, being the initial stage of microorganism growth curve, is not visible possibly due to a 5-hour incubation period of the sample. After the lag phase comes the log phase, also known as the stationary phase where E.coli growth decelerates. It remains uncertain whether cell division happens uniformly, some cells are dying off, or if the population simply stops growing and dividing.

During this phase, secondary metabolites such as antibiotics can be produced. The stationary phase occurs when there is a lack of nutrients or oxygen in the shake flask, or when cell reproduction is so rapid that both oxygen and nutrients become insufficient. In the subsequent glucose analysis test, a specific volume of sample, such as 2 ml, is transferred into a centrifuge tube and centrifuged for 10 minutes at 10,000 rpm. This separates the supernatant and biomass, and the supernatant is then used for glucose analysis.

The glucose sample is split into two portions, one that is diluted and one that is not, in order to compare their readings. The concentration of glucose (dextrose) is directly analyzed using the YSI 2700 turntable and a biochemical analyzer. Within the YSI dextrose membrane (YSI 2365), immobilized glucose oxidase is used for glucose analysis. According to Figures 2.1 and 2.2, it can be deduced that the E. coli colony consumed glucose as a nutrient for its growth, as indicated by the decreasing levels of glucose over time.

At high nutrient concentrations, the specific growth rate remains unaffected. However, at low concentrations, it strongly depends on the nutrient concentration. Although Monod anticipated this relationship, his equation does not accurately forecast it across all nutrient concentrations. If the equation’s parameters are estimated using results from low concentrations, then the specific growth rate at high concentrations exceeds what Monod’s equation predicts.

These findings were based on the assumption that the growth rate is influenced by multiple parallel reactions, each facilitated by enzymes with varying affinities. The final assessment involves measuring the cell dry weight by subjecting the sample to centrifugation and separating it into supernatant and biomass. The glucose analysis is then conducted using the supernatant, while the weight of the biomass is measured to determine the mass of the E. coli colony.

The biomass of E. coli undergoes a process of oven drying to remove all water content. Following this, the biomass is placed in a dissicator to cool down before being weighed using an analytical balance. The graph presented in figure 2.3 displays the cell dry weight, indicating that the biomass of E. coli is not consistently stable. This may be attributed to experimental errors that occurred during the process. It is assumed that the cell dry weight of E. coli follows the growth curve observed in the theory section for microorganisms.

The biomass of E. coli should increase over time as the cell number increases during binary fission. The study also measured the relationship between specific growth rate and mean cell volume. The results suggest that the mean cell volume is influenced by both the specific growth rate and the type of limiting nutrient. Different limiting nutrients can result in different mean cell volumes, even at the same specific growth rate. Therefore, the specific growth rate does not solely determine the mean cell volume.

Conclusion

From the analysis of the growth kinetic curve of E. coli, it can be concluded that the experiment was successful. This conclusion is supported by the similarity between the optical density graph and the growth curve of microorganisms. However, there was an error in measuring the cell dry weight, leading to unstable biomass readings. The glucose analysis showed that all glucose was consumed by the E. coli colony. Overall, studying the growth curve of microorganisms suggests that E. coli growth is complete in 24 hours using a shake flask.

Recommendation

In future experiments, it is recommended to weigh the mass of an empty cuvette before inserting the sample. This is important because not all cuvettes have the same weight, and there may be variations in weight between the second cuvette and the first cuvette. Additionally, it is necessary to consider the weight of both the sample and the cuvette for a more accurate analysis. It is also important to practice aseptic technique when transferring cultures to prevent the introduction and presence of other microorganisms in the culture media.

It is advised to use a different or larger cuvette for inserting the 2ml of culture media. Using a small cuvette may result in the cuvette being unable to store it. When using the spectrophotometer, it is important to take a reading of the empty medium without E.coli before taking a reading of the culture medium.

References

1. Growth and Vulturing Bactering, 24. 3. 2012 from url http://www. mansfield. ohio-state. edu/~sabedon/black06. htm
2. Microbial Growth Curve Part 2:Stationary PhaseFacts andFallacies,24. . 2012 from url http://fermentationtechnology. blogspot. com/2010/01/stationary-phase-facts-and-fallacies. html
3. Talaat E. Shehata and Allen G. Marr, Effect of Nutrient Concentration on the Growth of Escherichia coli (1971) Department of Bacteriology, University of California, Davis, California 95616
4. Roger G. Harrison,Paul todd,Bioseparation science and engineering(2003),oxford university press.
5. Christie john geankoplis, transport processes and separation process principles (fourth ed),pearson prentice hall (2003)