Early stages of biofilm formation of the facultative thermophilic sporeforming Bacillus licheniformis on stainless steel surface

Abstract

Bacteria can live in a surface attached community called biofilm, where microorganisms can multiply, grow and produce extracellular polymeric substances (EPS) and biofilm formation proceeds in different stages from attachment to a mature biofilm. In food industry, biofilms are a significant issue that can affect product quality and safety when biofilms are formed in the processing equipment.

The main aims of this study were to develop a method to study the early stages of biofilm formation in a laboratory and to determine how long it takes for the different stages of biofilm to form. The purpose was to simulate the situation in dairy processing systems by choosing a bacterial strain commonly found in milk and use continuous flow. Biofilm formation is dependent on many variables and all of those affect the timescale. Variables under investigation in this study included the media and surface material.

Bacillus licheniformis was used as model organism and biofilms were grown in a biofilm reactor on stainless steel or borosilicate glass surface material. The obtained sample coupons were investigated under scanning electron microscope and with fluorescent staining protocol. As an outcome, the developed method can be successfully used for biofilm studies and as a conclusion it could be stated that B. licheniformis is capable of forming a biofilm as a mono-culture. In a laboratory setting with continuous flow of milk, attachment of cells was detected ≤24 hours and mature biofilm in samples after 48 hours. EPS production was identified in samples collected after 32 hours and onwards.

Popular Abstract

Biofilms – challenge for dairy product quality

Bacterial communities, called biofilms, are a natural phenomenon but extremely unwanted in dairy industry. More has to be known about them in order to fight against these contamination sources and prevent them from spoiling product quality.
Bacteria can live in surface attached communities, where they multiply, grow and produce slimy substances in order to stick together. These biofilm communities are natural and present everywhere. Like the slime that appears in a flower vase or the yellow dental plague on your teeth are examples of natural biofilms that are always there. Biofilms can be beneficial and are essential for bacteria, but in dairy processing equipments they are problematic. If biofilm gets a chance to grow in dairy equipment it can release bacteria to the product stream and find its way to a ready product. Contaminated product might have off-flavor and –smell, become disgusting or, in worst case, be dangerous to health.

Biofilm formation goes through a series of steps before it is mature. In the start, bacteria must come in contact with a surface and be able to attach to it. After the bacteria have firmly attached, they start to replicate and produce extracellular polymeric substances (EPS) – the slimy substances. The purpose of EPS is to hold cells together and enable functionality of the community as well as to protect the bacteria. The more the bacteria multiply, the more biofilm develops and grows in size. Mature biofilm can have three-dimensional form and harbor millions of bacteria cells covered with protective EPS layer.
Food industry has an interest to prevent and control the growth of biofilms in processing equipment and better understanding of how biofilms are formed would help to avoid them. Therefore biofilm was chosen as a topic for this study. The study focused on developing a method to do research on early stages of biofilm formation in a laboratory. For this purpose, biofilms were grown in a small scale biofilm reactor and formation was followed with microscopic and staining methods.

Main aims of the study were to develop a method to perform the research and to estimate a timeframe of how fast biofilm formation happens. A challenging part is that the biofilm formation is very dependent on several different variables, such as temperature and surface material, and therefore the conditions influence the results. To conclude it was found that the specific bacterial species Bacillus licheniformis can form a biofilm, and with optimal growth temperature, mature biofilm formation takes 48 hours in a continuous flow of milk. This information can be used when designing dairy processes, including also frequency of cleaning and type of detergent, and the developed method can be used in the future studies.

• author: Kemppainen, Aulikki
• supervisor: Jenny Schelin; Peter Rådström; Birgitta Svensson
• organization: Applied Microbiology; Biotechnology
• course: KMBM01 20152
• year: 2016
• type: H2 - Master's Degree (Two Years)
• subject: Technology and Engineering; Agriculture and Food Sciences
• keywords: spores, Stainless steel, Bacillus licheniformis, Biofilm, Dairy processing, SEM, applied microbiology, teknisk mikrobiologi
• language: English
• id: 8881137
• date added to LUP: 2016-06-15 13:16:08
• date last changed: 2020-01-24 13:23:57