1

Large scale microbial fermentationprinciple and its problems

Numair Ahmad24

Indus International Institute

2

What is fermentation?

• Pasteur’s definition: “life without air”, anaerobe red ox reactions in organisms

• New definition: a form of metabolism in which the end products could be further oxidized

For example: a yeast cell obtains 2 molecules of ATP per molecule of glucose when it ferments it to ethanol

3

What is fermentation techniques (1)?

Techniques for large-scale production of microbial products. It must both provide an optimum environment for the microbial synthesis of the desired product and be economically feasible on a large scale. They can be divided into surface (emersion) and submersion techniques. The latter may be run in batch, fed batch, continuous reactors

In the surface techniques, the microorganisms are cultivated on the surface of a liquid or solid substrate. These techniques are very complicated and rarely used in industry

4

What is fermentation techniques (2)?

In the submersion processes, the microorganisms grow in a liquid medium. Except in traditional beer and wine fermentation, the medium is held in fermenters and stirred to obtain a homogeneous distribution of cells and medium. Most processes are aerobic, and for these the medium must be vigorously aerated. All important industrial processes (production of biomass and protein, antibiotics, enzymes and sewage treatment) are carried out by submersion processes.

5

Some important fermentation products

Product Organism Use

Ethanol Saccharomyces cerevisiae

Industrial solvents, beverages

Glycerol Saccharomyces cerevisiae

Production of explosives

Lactic acid Lactobacillus bulgaricus

Food and pharmaceutical

Acetone and butanol

Clostridium acetobutylicum

Solvents

-amylase Bacillus subtilis Starch hydrolysis

6

Some important fermentation products

7

Some important fermentation products

8

Winemaking fermenter

9

General Aspects of Fermentation Processes

10

Fermenter and its types

The heart of the fermentation process is the fermenter. Simple fermenters (batch and continuous)

Fed batch fermenter

Air-lift or bubble fermenter

Cyclone column fermenter

Tower fermenter

Other more advanced systems, etc

The size is few liters (laboratory use) - >500 m3 (industrial applications)

11

Cross section of a fermenter for Penicillin production  

12

In the manufacture of penicillin, fermentation is the preferred route. The fermentation substrate consists mainly of the sucrose found in corn steep liquor and lactose together with minerals and phenylacetic acid. The preferred mold is Penicillium chrysogenum and the first stage of the process is the transfer of the mold to the fermenter from the test tube where it has been stored.

13

The whole process is aerobic (it requires air). The fermentation is carried out over 4 or 5 days at 25-27°C. This gives a yield of about 0.5% penicillin. The mycelium, the mold cells plus insoluble metabolites, is filtered off in a coated drum filter.

14

The filtrate is adjusted the pH 2 with sulphuric acid. At this pH, the penicillin exists as an undissociated acid; consequently, it is soluble in organic solvents. After extraction the solution is decolourised and impurities are removed by activated carbon in a second drum filter.

15

The penicillin is then precipitated by addition of a solution of potassium acetate. This converts the penicillin into a negative ion, which makes it insoluble in the organic solvent. Inn the final steps the penicillin is crystallized and dried.

16

Flow sheet of a multipurpose fermenter and its auxiliary equipment

17

Fermentation medium

• Define medium nutritional, hormonal, and substratum requirement of cells

• In most cases, the medium is independent of the bioreactor design and process parameters

• The type: complex and synthetic medium (mineral medium)

• Even small modifications in the medium could change cell line stability, product quality, yield, operational parameters, and downstream processing.

18

Medium composition

Fermentation medium consists of:

• Macronutrients (C, H, N, S, P, Mg sources water, sugars, lipid, amino acids, salt minerals)

• Micronutrients (trace elements/ metals, vitamins)

• Additional factors: growth factors, attachment proteins, transport proteins, etc)

For aerobic culture, oxygen is sparged

19

Inoculums

Incoculum is the substance/ cell culture that is introduced to the medium. The cell then grow in the medium, conducting metabolisms.

Inoculum is prepared for the inoculation before the fermentation starts.

It needs to be optimized for better performance:

• Adaptation in the medium

• Mutation (DNA recombinant, radiation, chemical addition)

Problems faced in microbial fermentation

20

Bacterial contamination

21

22

This is the most common type of contaminant in bacterial cultures. Depending on the type of bacterium contaminating the process, the effects brought about are different. In all cases, one may expect reduced yield of product, and possible contamination by metabolites of contaminating bacteria. In some cases toxins may be produced by contaminants.

23

The probability of occurrence of a particular type of contaminations caused by bacteria may differ, depending on the process itself and the organism used in the process. One of the most frequently occurring contaminants are lactic acid bacteria and sporulating bacteria. Clean-up and re-occurrence prevention of especially the latter presents a considerable challenge.

Virus contamination

24

25

Viruses are the most dangerous contaminant in this type of fermentation. Due to small sizes, they are the toughest enemy to prevent entering the process, and their occurrence may result in very long, lasting even several months, paralysis of whole facility productivity. Due to the fact that eukaryotic cell cultures are usually used for production of highly valuable products, a risk of running fermentation without optimal protection is something what can cost you a lot.

Fungal infection

26

27

Fungal cultures can be contaminated also by other fungi. As this type of contamination may be relatively hard to detect, good measures of precaution should be undertaken to prevent it.

Dairy contamination

28

29

In dairy industry, controlling contamination issues is very important, not only due to possible health hazards. Milk is a raw material, which cannot always be sterilized, and almost every batch contains bacteriophages infectious to bacteria used in production process.

30

Moreover, some phages attacking lactic acid bacteria are resistant to pasteurization, and thus, they are much harder to eliminate. As it is not always possible to prevent bacteriophages from entering production process, there is a possibility to prevent re-infections, which are the ones to blame for vast majority of production failures.