(1) Fermentation process of penicillin：
Bacteria contamination will seriously affect the production yield and quality of penicillin, because so much miscellaneous bacteria can produce penicillinase, the penicillin will be decomposed and destructed. Therefore, bacteria contamination should be strictly controlled in the early stage, middle stage and late stage of fermentation.
(2) Fermentation process of nucleoside or nucleotide：
Because the strains, which is used in fermentation production, are a variety of nutrient-deficient microorganisms, their growth ability is poor, and they require nutrient-rich culture medium , it is prone to be contaminated by miscellaneous bacteria. After the early stage and middle stage of fermentation, the nutritional ingredients in the culture medium are rapidly consumed, which seriously inhibits the growth of the producing strain and the generation of metabolites.
(3) Fermentation process of organic acids such as citric acid：
In general, the pH value of the fermentation liquid after acid production is relatively low, and the growth of miscellaneous bacteria is very difficult. It is not likely to occur bacteria contamination in the fermentation process of early stage and late stage, and the attention is mainly paid to prevent the bacteria contamination in the early stage of fermentation process.
(4) Fermentation of glutamate：
Fermentation cycle of glutamate acid is short, the producing strain reproduces fast. If the culture medium is not too rich, it is generally less likely to be polluted by bacteria. If the seed tank is contaminated with bacteria, there will be great harm , so the occurrence of seed contaminated by bacteria should be strictly controlled. Once the seeds are found to be contaminated by miscellaneous bacteria, they should be discarded after sterilization, and the seed tanks, pipes, etc., should be carefully inspected and the sterilization should be conducted thoroughly.
2.1. Bacteria contamination in seed culture stage
Seed culture is mainly the growth of propagating bacteria, the concentration of bacteria is low, the culture medium is rich in nutrients, which leads the result that it is easy to be contaminated by bacteria at this time. Seed contamination should be strictly controlled, when the seeds are found to be contaminated, they should be discarded after sterilization, and the seed tanks and pipes should be inspected and should be sterilized thoroughly. The influence of bacteria contamination on fermentation during seed culture process is mainly reflected in the following aspects:
(1)Metabolism is obstructed:If it is contaminated by bacteria in seed culture stage, it will lead to serious interference in the growth & reproduction of producing strain and the generation of metabolites. Miscellaneous bacteria will compete with producing strain for nutrients and oxygen, which will lead to the result that the growth of producing strain is hindered.
(2)Quality of seed declines:Bacteria contamination can lead to the result that quality of seed declines, as the growth of miscellaneous bacteria consumes nutrients in the culture medium, which causes the producing strain unable to obtain sufficient nutrients, and it further affects their performance during the fermentation process.
(3)Fermentation process is unstable:Bacterial contamination during seed culture stage will cause the fermentation process to become unstable, because the growth and reproduction of miscellaneous bacteria will change the physical and chemical properties of the fermentation liquid, such as the decline in pH value, the rapid consumption of sugar and nitrogen, etc., which will affect the metabolism of the producing strain and the generation of product.
(4)Quality of generated product declines :If the seed is contaminated by bacteria during culture stage, it will lead the negative impact on the quality of the product. For example, after filamentous bacteria fermentation is polluted during fermentation process, a lot of hypha autolysis, the fermentation liquid is tacky, and some fermentation liquid even smelly. It is difficult to filter the fermentation liquid, and it is more difficult to filter the bacteria in the early stage of fermentation, which seriously affects the extraction rate and quality of the product.
2.2.Bacteria contamination in the early stage of fermentation
In the early stage of fermentation, the bacteria mainly grow and reproduce, and the generation of metabolites is few. At this time, it is easy to be infected with bacteria. After contamination, the miscellaneous bacteria reproduce rapidly, and the miscellaneous bacteria compete with the producing strain for nutrients and oxygen, which seriously interfere with the growth and reproduction of the producing strain and the generation of products. It is necessary to prevent bacteria contamination in the early fermentation stage.When bacteria contamination occurs during the early stage of fermentation, if the nutrients are not seriously consumed, they should be quickly re-sterilized,and they should be supplemented with necessary nutrients, and re-inoculation for fermentation should be conducted. The influence on fermentation when bacteria contamination occurs during the early stage is mainly reflected in the following aspects:
(1)Amount of enzyme activity:Bacterial contamination during the early stage of fermentation will affect the quantity of enzymes. Miscellaneous bacteria reproduce rapidly, and it will consume the nutrients in the culture medium, then it will affect the reproduction of producing strain, and thus it will reduce the quantity of enzymes.
(2)Effect of enzyme application :Bacterial contamination during the early stage of fermentation will affect the application effect of enzyme. The miscellaneous bacteria will produce some metabolites, and these metabolites may competitively inhibit the enzyme of the producing bacteria, thus it will affect the application effect of enzyme.
(3)Production sequence:Bacteria contamination during the early stage of fermentation will affect the production sequence. The equipment needs to be sterilized after contamination, which will affect the production schedule and tank discharge plan. In addition, if the miscellaneous bacteria reproduce too much, it may lead to the negative affect on quality of the fermentation liquid.
2.3.Bacteria contamination during the middle stage of fermentation
Bacteria contamination during the middle stage of fermentation will seriously interfere with the metabolism of producing strain and affect the generation of products. Some miscellaneous bacteria produce acidic substances after reproduction, which will lead to the result that the pH value declines, the sugar and nitrogen is rapidly consumed , the autolysis of bacteria (filament) body occurs, the fermentation fluid is tacky, plenty of foams are produced, the accumulation of metabolites is rapidly reduced or stopped, some of the generated products will also be used or destroyed, and some of the fermentation liquid is smelly. The influence on fermentation when bacteria contamination occurs during the middle stage is mainly reflected in the following aspects:
(1)Interfering with metabolism:Bacteria contamination during the middle stage of fermentation will seriously interfere with the metabolism of producing strain. The miscellaneous bacteria produce a lot of acid, and it leads to the result that pH of the culture liquid declines, which may destroy the cell membrane and cell wall of the producing strain and may affect their growth and reproduction. At the same time, the consumption rate of sugar and nitrogen is accelerated, which leads to the result that fermentation liquid is tacky, autolysis of mycelium occurs, and secretion of generated product reduces or stops.
(2)Producing toxin :Some miscellaneous bacteria will produce toxins which may be toxic to the producing strain or even cause fermentation to fail. For example, escherichia coli contamination may cause the culture liquid to be black, smelly, and foamy in large amounts.
(3)Destructing generated products:Sometimes miscellaneous bacteria will use the generated products to reproduce, which will destroy the structure and function of the generated product and will cause the quality and quantity of the product to decline.
(4)Affecting later treatment process:If the fermentation liquid is seriously polluted by miscellaneous bacteria, it may lead to the negative impact on the later treatment process. For example, excessive proliferation of miscellaneous bacteria can make it difficult to conduct the filtration and affect the extraction and refining of products.
2.4.Bacteria contamination during the late stage of fermentation
Accumulation of generated product during the late stage of fermentation is relatively more, sugar and other nutrients will be exhausted. If the amount of bacteria contamination is not too much, the fermentation can be continued; If the contamination is serious and destructive, measures can be taken to conduct tank discharge in advance. The influence on fermentation when bacteria contamination occurs during the late stage is mainly reflected in the following aspects:
(1)Destructing generated products:During the late stage of fermentation, a large number of generated products, especially antibiotics, have accumulated in the fermentation liquid. These antibiotics have the certain inhibition or killing effect on miscellaneous bacteria. If bacteria contamination occurs at this stage, it may break down the products that have already generated,which will lead to the loss of generated products and decline of quality.
(2)The quality of fermentation liquid declines:During the late stage of fermentation,bacteria contamination will cause quality of the fermentation liquid to decline. The reproduction of miscellaneous bacteria will consume the nutrients in the culture medium, change the pH value, and produce some metabolites, which may have the destructive effect on the cell membrane and cell wall of the producing strain, then it will affect the quality and yield of the generated products.
(3)Increasing risk of bacteria contamination:During the late stage of fermentation, bacteria contamination may lead the fermentation liquid to be smelly, black and foamy. These phenomena may increase the risk of bacteria contamination and cause the fermentation process to be more unstable and uncontrollable.
During the fermentation process, miscellaneous bacteria contamination is a problem that needs to be paid close attention. Miscellaneous bacteria contamination may lead to the decline in the quality of fermentation products, decline in yield, and may even lead to the failure of the fermentation process. Therefore, the prevention and control of miscellaneous bacteria contamination is of great significance for the fermentation industry. The following content will discuss the prevention and control measures of fermentation bacteria contamination in the aspects of raw material control, sterilization treatment, inoculation purification, equipment cleaning, process monitoring, prevention of air pollution, strains preservation, waste water treatment and so on.
(1)Control of raw material
Control of raw material is the first line of defense to prevent miscellaneous bacteria contamination during fermentation process. Qualified raw material is the premise to ensure the smooth progress of the fermentation process. Therefore, raw materials must be strictly screened to ensure that they do not contain miscellaneous bacteria. At the same time, the storage and management of raw material is also crucial. It is necessary to ensure that the warehouse of raw material is clean and dry to avoid moisture, mildew and contamination of raw materials. For unqualified raw materials, they should be treated and replaced in time. In addition, the management of raw material suppliers should be strengthened to ensure the reliability of the raw material supply chain.
(2)sterilization treatment
Sterilization treatment is a key step to prevent the contamination of fermentation miscellaneous bacteria. Before fermentation begins, it is necessary to thoroughly sterilize the culture medium, fermentation equipment, pipes, etc. Common sterilization methods include dry heat sterilization, moist heat sterilization, gamma ray sterilization, etc. Choosing the proper sterilization method needs to consider the nature of the fermented product, heat resistance and other factors. At the same time, choosing the proper sanitizer is also an important part of sterilization treatment. Sanitizer should have the broad spectrum of antibacterial properties, which can effectively kill a variety of miscellaneous bacteria, but also should consider its residual effect. When using sanitizer, it is necessary to follow the guidelines for use to ensure the effectiveness and safety of the sanitizer.
(3)Inoculation and purification
Inoculation and purification is an important link to prevent the contamination of miscellaneous bacteria during fermentation process. Before inoculation, the strains need to be purified and screened to ensure that they do not contain miscellaneous bacteria. At the same time, it is also necessary to conduct thoroughly clean and disinfect the fermentation equipment, pipelines, etc., to avoid the residue of miscellaneous bacteria. During the inoculation process, the introduction of miscellaneous bacteria should be avoided, such as the use of sterile inoculation equipment and standardized operation procedures. After inoculation, the quality of the fermentation liquid needs to be tested, and measures should be taken to deal with the contamination of miscellaneous bacteria in time if contamination of miscellaneous is found.
(4)Equipment cleaning
Equipment cleaning is one of the important measures to prevent contamination of miscellaneous bacteria during fermentation process. Equipment cleaning includes the cleaning on surface of the equipment , sterilization and dust removal. Cleaning equipment can remove dirt, bacteria and other sundries on the surface of the equipment to avoid it becoming a breeding ground for miscellaneous bacteria. At the same time, it is also the key to choose the proper cleaning agent and disinfectant. Cleaning agents and disinfectants should have a broad spectrum of antibacterial properties,which can effectively kill a variety of miscellaneous bacteria, but also should consider their residual effects and safety. In the process of cleaning and disinfection, it is necessary to pay attention to the dead corners and gaps of the equipment and other easily ignored places to ensure the cleaning effect of the equipment.
(5)Process monitoring
Process monitoring is an important means to prevent contamination of miscellaneous bacteria during fermentation process. Process monitoring includes real-time monitoring and recording of temperature, humidity, pressure, gas composition, material flow and other parameters during the fermentation process, and timely discovery of miscellaneous bacteria pollution and other problems. At the same time, after the end of fermentation, the quality of the fermentation liquid needs to be tested, if miscellaneous bacteria contamination and other problems are found, it is necessary to implement timely analysis and summary of the causes, so as to take measures to conduct the improvement.
(6)Preventing air pollution
Air is one of the important ways for the spread of miscellaneous bacteria. In order to prevent air pollution, a series of measures need to be taken. Firstly, the source of pollution should be controlled and raw materials & equipment containing miscellaneous bacteria should be avoided. Secondly, ventilation should be strengthened to keep clean and dry of the fermentation workshop , so as to avoid the influence of humidity and dust in the air during fermentation process. In addition, methods such as air filters can also be used to remove miscellaneous bacteria and dust in the air, so as to reduce air pollution during fermentation process.
(7)Strains preservation
Strains preservation is one of the important measures to prevent the contamination of miscellaneous bacteria. Selecting the appropriate preservation conditions and methods can prolong the shelf life of the strains and prevent the contamination of miscellaneous bacteria. The commonly used preservation methods include glycerin preservation, vacuum drying preservation, liquid nitrogen preservation, etc. In the preservation process, it is necessary to pay attention to the control of temperature, humidity, oxygen and other parameters to ensure the safety and stability of the strains. At the same time, the quality of the strains should be tested regularly. If problems such as miscellaneous bacteria contamination are found, it is necessary to adopt timely measures to deal with these problems.
(8)Waste water treatment
Waste water treatment is one of the important steps which prevent bacteria contamination in fermentation industry. Waste water treatment should follow strict waste water treatment standards to ensure that the discharged waste water meets the requirements of environmental protection. Commonly used waste water treatment methods include physical methods, chemical methods and biological methods. When choosing the waste water treatment method, factors such as the nature and composition of the waste water need to be considered. At the same time, the choice of waste water treatment equipment is also crucial. The equipment should have the characteristics of high efficiency, stability and durability, which can effectively remove miscellaneous bacteria and harmful substances in the waste water, so as to ensure the effect and reliability of waste water treatment.