Title: 2.3 KSB III: Monitoring Bioreactor Growth and Products
Course: ATEP - Biotechnology Module B > Section: Section 2 Research and Investigate

2.3.1 KSB III

KSB III

How will you know your bacteria are growing, product is being made and contamination is minimized?

Once you have successfully grown your culture, it will be time to take some data.

As you move through this section, complete the worksheet titled: Knowledge and SkillBuilder III: Measurements for the Bioreactor That Could Affect the Design of the Bioreactor.

You may need to return to Module A lessons or the Technology Park to review.

If you are not clear about an answer, use the Internet to research. Generally, a website that ends in an .edu, .org or .gov can be considered a valid source.

Then check your research with your instructor.

2.3.2 Monitoring Growth

There are three ways to monitor growth: visual, plating on agar and the use of a spectrophotometer.

Visual: The more growth there is, the more cloudy, or turbid your culture will be. This of course produces no quantitative data and there is no way to know exactly what is growing, but it can be used as an initial observations to show that your media is supporting some type of growth.

Plating on agar: A sample of your culture can be plated on and agar plate and colonies counted over time to identify growth. To use this method you will first have to have a positive control, know what lactobacillus looks like when grown on an agar plate known as the colonies morphology.  Plating a pure lactobacillus sample can do this.

Spectrophotometer: Culture sample an b placed in a spectrophotometer to obtain absorbance readings based on the turbidity of the solution. This is the best technique to monitor growth.

2.3.3 Monitoring Contamination

It will be important to ensure that your culture is pure from beginning to end. If other bacteria are introduced into the bioreactor at any time, your results will be invalid. The presence of contaminants may affect the growth of your organism and the desired product. Also, there is no way to tell where the protease originated from if multiple organisms are being grown in the bioreactor.

Plating

To monitor contamination, you will grow the culture in a Petri dish containing media specially designed to promote the growth of your organism. If there is no growth, this may be an indicator that your bacteria is dead. If your plate shows multiple types of colonies, contamination can be confirmed.

The plate on the left contains only one type of colony. The plate on the right contains contamination.

2.3.4 Monitoring Protease Production

You will use a gelatin assay to monitor protease production. Here is how it works:

Protease breaks down proteins. Gelatin is a protein. So adding protease to gelatin will result in the breaking down of the gelatin protein into smaller units. When this happens, the gelatin is liquefied. The more liquid, the higher the protease concentration. Creating a control will be very important in making sure your data is precise.

Protease is found in meat tenderizers and should be tested using a constant amount of gelatin. How will you measure the amount of liquid produced? How fast will this reaction occur? These are parameters that you will decide once you have begun your testing.