Category: Contamination

Fermentation for Everyone

Fermentation – what a broad topic. People think that most fermentation types are different from each other and they really are not. Fermentation is the production of a product in a tank by usually a mono culture (but not always – Belgium ales, kombucha, etc) be it, fungi, bacteria, algae or yeast. Some have unique aspects, like totally anaerobic fermentation, which is really susceptible to oxygen. But, if you know the particulars of the organism, the basic process is not that different.

The big trick, with most fermentation processes are understanding the what the product is and what parameters (micro-organism biochemistry) are needed to produce it. These are considered the parameters of the process. For example, there are many bacterial enzymes, such as alpha-amylase, which are produced by starving the culture for carbon (discussed in papers such as Regulatory factors affecting alpha-amylase production in bacillus licheniformis. ). Knowing the fermentation parameters is important as is what was discussed in 3 previous posts, cleanliness and contamination – B. cereus about contamination – part 1.

An example of dealing with process parameters:  long fermentation times can have issues with contamination.  The longer the process time, the more susceptible the process is. This is where understanding the process and the micro-organism’s characteristics can help define the proper parameters. For example, I performed a 7 day fermentation to produce an enzyme I was interested in studying. The fungus I was studying was grown at a low pH (~pH4.0) to help prevent contamination. Since I understood the growth and  biochemical characteristics of the organism in question, it was easy to determine the best way to alleviate possible contamination and this was by lowering the pH of the initial fermentation broth. This helped to relieve any possible contamination issues. This is just one example of how understanding the parameters can bring you to a successful fermentation.

If you need help with fermentation design, scale-up, or contamination issues, GeoMetrick Enterprises can help.

David Slomczynski, Ph.D; GeoMetrick Enterprises

B. cereus about contamination – Part 3

In part 2  (  ) I covered problems with equipment. This post will cover cleaning. As was once told me by my graduate advisor, ‘Cleanliness is next to Godliness’. So, protocols for cleaning are important. Too many times people get accustomed to CIP (Clean In Place) but do not check whether or not the procedure actually works.

There are many tools to check for issues, one is from Hygienia and there are many others. This is a basic test to determine if there are intact bacteria, mold, etc. Then further tests, like culturing, etc. will determine what the microorganism is and what to do about it.

At one particular job, I was working on a project where sterility was important, since we were doing a continuous fermentation to a slow growing culture. It was important that the initial sterility and cleanliness were paramount. All the issues with dead spot, etc. were in place but how to clean was not discussed. We started with a CIP (Clean In Place) system but determined that the system caused issues, namely some Bacillus sp. that was found in the CIP (Clean In Place) material. We had to switch and tried several different vendors until one solution was found of a different make up.

In another instance, a simple brush was found to be the culprit in scoring the inside of a fermentor. It was these scratches, that allowed contamination to build up. We had dismantle the fermentor and polish the inside to remove these scratches before continuing with our process.

Cleaning protocols are not infallible and need to be tested to make sure they work properly.

All 3 posts show how important being observant and diligent in dealing with contamination can be.

David Slomczynski, Ph.D; Geometrick Enterprises

B. cereus about contamination – Part 2

In my previous post (   ), I covered the first on three main types of contamination. I will continue with number 2 in this post.

2. Dead spots in the process loop. This is a very common problem with fermentors and other vessels of similar design. It can also affect other process equipment as well, like ultrafiltration skids, etc. In all cases, areas of slow or no movement can be areas where contamination will build up. An example is a 100 liter sterilized in place fermentor I was working on. The agitation was from the bottom and had a live steam seal (steam is needed to keep the seal in place). Well, there were some small grooves in this seal and the seal (made if high temperature rubber) acted as insulation. This allowed some material to build up and and cause  partial contamination of some broths I was working on. Eventually, I talked to the equipment manufacturer who came up with a new seal design and installed it.

It is important if there is contamination, even a small amount, to evaluate all equipment to discern where it comes from.

In part 3, I will talk about cleaning procedures.

David Slomczynski, Ph.D; Geometrick Enterprises

B. cereus about contamination – part 1

One thing is known, you either admit you have contamination problems or you can bury your head in the sand. It is that simple. Contamination can be minimized and kept to a minimum as long as you know it is there. Many companies won’t admit it and that is a problem. Since this is a big topic I am planning to split this into 3 posts.

Contamination can come from a variety of sources. Some off the top of my head are: 

  1. Raw Material 
  2. Dead spots in the process loop 
  3. Bad cleaning procedures

I will explain some examples and what was done in those instances.

1. In graduate school, we were working with a fungus where we would grow it on a semisolid medium with shredded wheat as the solid support. Well, the first time we made the medium and let it sit for a day, we had bacterial contamination on the medium. As it turns out, there was a Bacillus sp. that contaminated the shredded wheat in very minute quantities. When we sterilized the shredded wheat, it helped the bacterium to grow by reactivating the spores present. To alleviate this issue, we used a process called Tyndallization. Basically, we sterilized the material twice with a 1/2 day pause in between. This allowed the spores present to germinate and then we were able to kill them during the 2nd sterilization step.

I will cover contamination types 2 and 3 in my next 2 posts.

David Slomczynski, Ph.D; Geometrick Enterprises