Opening the book…
- Perform aseptic techniques while streaking or inoculating bacteria onto starch plates to prevent contamination.
- Understand how starch hydrolysis is connected to the amylase enzyme in bacteria.
- Interpret the results of the starch test based on the color changes of bacterial colonies after iodine is introduced into the media.
Starch Plates
Starch plates are a differential media used to determine if bacteria can hydrolyze starch using the enzyme amylase. While starch plates can be used to determine if any bacterial species has the amylase enzyme, this media is often used to differentiate between Clostridium and Bacillus species.
The main chemical component of a starch plate is starch, a complex polysaccharide made up of glucose molecules connected together in a long, branch-like pattern. Starch is an excellent energy source for bacteria, but the long branching chains of the starch molecule make it too big to pass through the bacterial cytoplasmic membrane. Bacteria have to break down or hydrolyze starch into single units of glucose, called monosaccharides, using the enzyme amylase. Glucose can easily pass through the bacteria's membrane and then be used to produce energy. Bacteria capable of breaking down starch have an advantage in energy production as starch is often readily available in the bacteria's natural environment.
Figure 1. The complex molecule starch is broken down by the bacterial enzyme amylase forming single sugar molecules (glucose monosaccharides) and double sugar molecules (maltose disaccharides) Glucose molecules are easily transported across the bacterial cytoplasmic membrane due to their small molecular size. Once inside the cell, glucose can be used to produce vast amounts of energy for the bacteria.
The bacterial enzyme amylase is defined as an exoenzyme. Exoenzymes are excreted by the bacteria into their surrounding environment. Thus, when bacteria are in environments with high quantities of starch (like starch plates), the cells will start to produce and secrete amylase into their surroundings. Once amylase is secreted into a starch plate, it will start to break down the media, creating areas of clearing around the bacterial colonies. Starch hydrolysis on starch plates is invisible, however, and iodine must be added to the plates after incubation to determine if bacteria are producing amylase. Iodine will form a strong bond or complex with starch molecules that have not been broken down. This bond turns the media black wherever starch is present. If iodine is added to a starch plate and the entire media surface (including the bacterial colonies) turns black, the bacteria are incapable of breaking down starch and do not produce the amylase enzyme. If bacteria are producing amylase and breaking starch down, the iodine has nothing to bond with and will leave the plate clear, creating halos around the bacteria.
Figure 2. Starch hydrolysis on starch plates is not visible without the addition of iodine. After adding iodine to the media, bacteria that can produce the amylase enzyme to break down starch molecules will have clear halos surrounding colony growth. Bacteria that can not produce amylase will not alter the starch content of the media and the plate and colony growth will all turn black in color.
Commercial Uses Of Microbial Amylase Enzymes
Microbial amylase enzyme production can be genetically modified to support variable industries and medical science. There are three different types of amylase enzyme: α-amylase, β-amylase, and γ-amylase. The α-amylase, also called alpha-amylase, enzyme is the most widely used form for commercial enzyme applications.
As such, large quantities of pure alpha-amylase are sought after by many industries. Alpha-amylase is produced by fungi, plants, bacteria, and many other organisms. However, bacteria have been found to have the fastest production of high volumes of the enzyme. Further, bacteria can be easily genetically manipulated to add additional characteristics to the enzyme including thermal stability, extreme cold tolerance, and extreme salt tolerance. The most common industrial bacteria used for alpha-amylase production include: Bacillus stearothermophilus, Bacillus amyloliquefaciens, and Bacillus licheniformis.
Alpha-amylase produced by Bacillus species is most often used to form detergents. Typical laundry and dish detergents contain chemicals that pollute waste-water as they are not easily broken down by typical waster-water cleaning procedures. This polluted water can enter rivers and other water bodies eventually leading to excessive microbial organism growth and depleted water oxygen levels. The addition of alpha-amylase to detergents removes these harsh chemicals while also enhancing the detergents ability to break down tough stains, which typically contain starch. Alpha-amylase has many other applications including:
- Fuel alcohol (ethanol) production.
- Food production: bread, flour, juice, beer, and more.
- Yarn, paper, and other textiles production.
Thus, large scale production of alpha-amylase is crucial for many industries. New applications for alpha-amylase and new microbes that produce the enzyme are still being studied. Future applications of the enzyme could focus on energy conservation and increased enzyme stability through new protein engineering procedures. Overall, the amylase enzyme is a key player in the success of human commercial goods production.
Attributions
"Microbiology Laboratory Manual: Labs, 1.17 Starch Hydrolysis" by Dr. Rosanna Hartline , LibreTexts: Biology, West Hills College Lemoore is licensed under CC BY-NC-SA 4.0
"Microbiology Textbook: Chapter 7, Microbial Biochemistry" by Openstax, Digital ISBN 13: 978-1-947172-23-4 is licensed under CC BY 4.0
"Microbiology Textbook: Chapter 8, Microbial Metabolism" by Openstax, Digital ISBN 13: 978-1-947172-23-4 is licensed under CC BY 4.0
Elyasi Far, B., Ahmadi, Y., Yari Khosroshahi, A., & Dilmaghani, A. (2020). Microbial Alpha-Amylase Production: Progress, Challenges and Perspectives. Advanced pharmaceutical bulletin, 10(3), 350–358. https://doi.org/10.34172/apb.2020.043 "This is an Open Access article distributed under the terms of the Creative Commons Attribution (CC BY)." and is licensed under CC BY 4.0
Image Citations
Figure 1
- "Microbiology Laboratory Manual: Labs, 1.17 Starch Hydrolysis" by Dr. Rosanna Hartline , LibreTexts: Biology, West Hills College Lemoore is licensed under CC BY-NC-SA 4.0
Figure 2, Modified From:
- "File:Starch hydrolysis plate.jpg" by RosieScienceGal, Wikimedia Commons is licensed under CC BY-SA 4.0
10.17: Starch Test is shared under a not declared license and was authored, remixed, and/or curated by LibreTexts.
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