How to analyze and take preventive measures based on the infection situation?

1. What are the possible ways to cause fermentation contamination?

Improper operation during strain cultivation

Incomplete sterilization of the culture medium

Poor sealing of fermentation equipment (such as fermentation tanks and pipelines)

Insufficient air sterilization

2. Since contamination is unavoidable, what should we do?

We need to improve production technology levels, strengthen management of the production process, and control the various stages prone to contamination to prevent fermentation contamination as much as possible; moreover, once contamination occurs, we should quickly identify the cause of the contamination and take corresponding effective measures to minimize the losses caused by fermentation contamination.

3. What are the types of contaminants that pose the greatest threat in different fermentation processes?

Penicillin fermentation: Clostridium butyricum

Streptomycin fermentation: Bacillus subtilis, Pseudomonas aeruginosa

Tetracycline fermentation: Diplococcus, Bacillus cereus, Capsulated bacillus

Glutamic acid fermentation: Bacteriophage

Citric acid fermentation: Penicillium

4. The impact of contamination at different production stages on fermentation:

Contamination during the seed cultivation period: Extremely harmful to the entire fermentation process

Contamination in the early fermentation stage: Severely interferes with the growth and reproduction of the production strain

Contamination in the mid-fermentation stage: Interferes with the metabolism of the production strain, affecting product generation

Contamination in the late fermentation stage: Relatively minor impact

5. The impact of different causes of contamination on fermentation:

Contaminated seeds: Will lead to an expanding range of contamination, causing significant losses to production.

Airborne contamination: Causes widespread contamination in fermentation

Incomplete sterilization of culture medium or equipment: Generally does not have continuity, leading to failure of individual (batch) fermentation tanks

Equipment leakage: Higher probability of contamination

6. Analysis of abnormal fermentation phenomena and causes of contamination:

(1) Abnormal changes in dissolved oxygen

When the contaminating bacteria are aerobic microorganisms, the dissolved oxygen decreases rapidly within a short time, approaching zero, and cannot recover for a long time;

When the contaminating bacteria are anaerobic microorganisms, and the production strain is inhibited from growing due to contamination, the oxygen consumption decreases, leading to an increase in dissolved oxygen.

(2) Abnormal changes in CO2 during exhaust

For example, when contaminated by foreign bacteria, sugar consumption accelerates, CO2 content increases; after bacteriophage contamination, sugar consumption slows down, and CO2 content decreases. Therefore, abnormal changes in CO2 content can be used to judge contamination.

(3) Other abnormal phenomena:

Such as poor growth of microbial cells, abnormal changes in pH, abnormal increase in foam during fermentation, abnormal color changes in the fermentation liquid, abnormal decrease in the content of metabolic products, prolonged fermentation cycles, and abnormal increases in the viscosity of the fermentation liquid can indicate contamination.

7. Analysis of contamination causes:

(1) Analyzing contamination causes based on the scale of contamination

Contamination in large fermentation tanks: Air system

Contamination in some fermentation tanks (or tank groups): Initially may be due to contaminated seeds or incomplete sterilization, while in the mid to late stages, it may be caused by issues in the intermediate feeding system or oil pipeline system.

Continuous contamination in individual fermentation tanks: Equipment issues (such as valve leakage or tank corrosion and wear), contamination caused by equipment corrosion and wear will show a trend of advancing contamination time for each batch of fermentation.

Occasional contamination in individual fermentation tanks: The reasons are relatively complex, as various contamination pathways may be involved.

(2) Analyzing based on the timing of contamination

Early fermentation contamination: Contaminated seeds, incomplete sterilization of culture medium and equipment, dead corners in equipment or pipelines.

Mid to late stage contamination: Intermediate feeding, equipment leakage, unreasonable operation.

(3) Analyzing based on the type of contamination:

Heat-resistant spore-forming bacteria: Dead corners or incomplete sterilization

Cocci, yeast: May be brought in from condensate of steam or air.

Light green colonies (Gram-negative bacilli): Leakage from the cooling pipes or jackets of fermentation tanks.

Molds: Incomplete sterilization or non-strict aseptic operation.

Reasons for equipment leakage: When equipment leaks, the time of contamination occurrence for each batch tends to advance.

a. Corrosive substances in fermentation liquid corroding the equipment

b. Abrasion

c. Poor equipment processing

Preventing leakage from coil pipes:

Carefully clean the fermentation tank after emptying.

Control the quality of cooling water, reducing the chloride ion content.

Regularly inspect and test for leaks in the coil, promptly identifying any gaps.

Leak testing methods: Air pressure test, water pressure test.

Preventing leakage from air distribution pipes.

④ Preventing leakage from the tank body.

⑤ Preventing leakage from fittings.

(4) Connection of pipelines:

Thread connection, flange connection, welding

(5) Reasons related to the air filtration system:

Cause analysis: air inlet, production environment, air filter

Contamination characteristics: The air filtration system carrying bacteria can cause contamination in every batch of fermentation tanks; at this point, a sterile sample test of the air filtration system must be conducted.

8. Inspection and judgment of contamination

Sterility test methods

Microscopic examination method (microscope inspection method)

Broth culture method

Plate streak culture or slant culture inspection method

Observation method for abnormal phenomena during fermentation process

If the broth shows a color change reaction (from red to yellow) or becomes turbid for three consecutive times, or if abnormal colonies are found in plate cultures for three consecutive times, it can be judged as contamination.

9. Rescue and treatment of mixed bacterial contamination:

(1) Contamination during seed culture period: should be discarded after sterilization, and the seed tank, pipelines, etc. should be carefully checked and thoroughly sterilized.

(2) Contamination in the early fermentation stage: since the nutrient components are not consumed much, rapid re-sterilization should be performed; additionally, necessary nutrient components should be supplemented, and re-inoculation for fermentation should be conducted.

(3) Contamination during mid to late fermentation: appropriate sterilants or antibiotics can be added along with normal fermentation liquid to inhibit the growth rate of mixed bacteria; if the product content reaches a certain value, it can be canned as long as it is confirmed to be contaminated.

(4) Treatment of equipment after fermentation: empty tanks should be heated and sterilized above 120℃ for 30 minutes before use. Formaldehyde fumigation or soaking in formaldehyde solution for more than 12 hours can also be used for treatment.

10. Pathways and prevention of mixed bacterial contamination:

(1) Seed carrying bacteria and its prevention:

Cause analysis: seed storage, seed culture equipment, seed replacement and inoculation, seed culture medium

Prevention measures:

Check the preserved strains used in the contamination.

Check the triangular flasks and other utensils used for inoculation in the contaminated seed tank.

Check the sterilization operations performed on triangular flasks, cotton plugs, culture media, etc.

Check the working condition of the sterilizer, calibrate the thermometer and pressure gauge. Ensure that the air inside the sterilizer is completely exhausted during operation.

Check the sterile conditions of the sterile room, especially the inoculation box. Check the sterilizing agents used for the inoculation box.

Check the operational skills of the personnel.

Conduct microbiological tests on mixed bacteria or bacteriophages.

(2) Airborne bacteria and its prevention:

Prevention measures:

Oil entering the air: switch to an oil-free lubricated compressor.

Perforation of tube-type coolers.

Loose filtering medium, causing air to short-circuit.

Filtering medium wet with water.

Aging of filtering medium.

(3) Contamination caused by equipment leakage or "dead corners" and its prevention:

Equipment leakage mainly refers to fermentation tanks, sugar supplement tanks, cooling coils, pipeline valves, etc., which can develop small leaks due to chemical corrosion, electrochemical corrosion, wear between metal and sand in raw materials, and poor processing.

"Dead corners" include certain parts of fermentation equipment or connecting pipelines and certain parts of culture media or other materials.

Leakage contamination of coils and prevention:

Cause analysis: due to temperature differences (internal cooling water temperature, external sterilization temperature), rapid temperature changes, or low pH of fermentation liquid, severe chemical corrosion, etc.

Prevention measures: careful cleaning, checking for leaks, or reducing the Cl-1 content in cooling water can be taken.

Contamination of "dead corners" in air distribution pipes and prevention:

Cause analysis: prone to wear and perforation due to stirring and airflow.

Prevention measures: frequent replacement of air distribution pipes or thorough washing.

Leakage contamination of fermentation tanks and prevention:

Cause analysis:

Localized chemical corrosion or wear occurs, leading to perforation and leakage.

Dirt tends to accumulate around welding points of the tank.

Poor manufacturing of fermentation tanks.

Various interfaces on the fermentation tank head.

Improper repair welding positions of fermentation tanks.

Prevention measures:

Applying anti-corrosion coatings on the inner wall of the tank, enhancing cleaning, and regularly removing dirt are effective measures to eliminate contamination.

Using stainless steel or composite steel can effectively overcome this drawback, and attention to cleaning can avoid contamination.

(4) Unreasonable installation or configuration of pipelines forming "dead corners":

Cause analysis:

Frequently gathering some pipelines into a main pipeline. Most pipelines use flange connections.

Prevention measures:

Using separate exhaust, drainage, and sewage pipes can effectively prevent contamination.

The processing, welding, and installation of flanges must meet sterilization requirements to minimize or eliminate measures such as connecting flanges.

⑤ Leakage of pipe fittings leading to contamination and prevention:

(4) Contamination caused by incomplete sterilization of raw materials:

*Cause analysis:

Starch-based raw materials are prone to clumping when heated too quickly or mixed unevenly, causing the center of the clumps to be "undercooked," making it difficult for steam to penetrate and kill contaminants.

The high solid content in media such as bran and soybean meal can splash onto the tank walls or various supports during loading, leading to accumulation. These accumulations have slower heat transfer during sterilization, making it difficult to kill some contaminants.

*Prevention and control measures:

Before heating, mix evenly by stirring and add a certain amount of amylase for liquefaction; if large particles are present, they should be screened out before sterilization.

For media with high solid content such as bran and soybean meal, pre-mixing outside the tank and then transferring to the fermentation tank for sterilization is more effective.

*Specific measures:

Raw materials purchased should be dry, and stored raw materials should be kept dry.

When preparing media, crush raw materials first with cold water to prevent clumping during mixing.

Crushed raw material particles should be small enough; large particles can affect sterilization effectiveness.

④ If the raw materials are severely contaminated with spores, after making the media, they can be kept at 30°C for several hours until the spores germinate.

Then proceed with sterilization.

(5) Contamination caused by improper operation and its prevention:

*Cause analysis:

During sterilization, due to unreasonable operation, the air inside the tank was not completely expelled, causing the pressure gauge to show "false pressure."

When foaming occurs severely, the foam can rise to the top of the tank or even escape, causing the foam to top the tank.

During continuous sterilization, the temperature and retention time of the media sterilization did not meet sterilization requirements.

④ Improper sterilization methods for probes or components.

*Prevention and control measures:

Open the exhaust valve before sterilization to expel cold air.

Add defoamer for foam treatment.

During continuous sterilization, avoid excessive fluctuations in steam pressure and strictly control sterilization temperature.

Ensure thorough sterilization of probes or components.

(6) Bacteriophage contamination and its prevention:

Prevention measures:

Carefully preserve production strains to ensure they are not contaminated.

Discharge of live bacteria is strictly prohibited.

Strengthen equipment management.

Enhance environmental sanitation work.

Strengthen monitoring of sterile air, environmental contaminants, and bacteriophages.

*Methods used after bacteriophage infection:

① Select resistant strains.

② Rotate the use of strains with different specificities.

③ Add chemical agents (e.g., during glutamic acid fermentation, add 2-4 ppm chloramphenicol, 0.1% sodium tripolyphosphate, 0.6% sodium citrate or ammonium, etc.).

④ Re-sterilize the culture solution and then re-inoculate (bacteriophages are not heat-resistant; 70-80˚C for 5 minutes can kill them).

⑤ Other methods.