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Microbial Fermentation of L-lysine: Introduction, Stages, Process, and Uses: 

Introduction to Lysine Production: 

• Lysine is an alpha amino acid used for the biosynthesis of Proteins.
• Lysine is an essential amino acid that can not be synthesized in humans so it must be taken through a balanced diet. 
• The sources of lysine include eggs, meat, fish, and dairy products.
• Lysine plays a key role in the human body such as it plays an important role in the formation of collagen etc.
• Lysine can produce in two ways Fermentation method and the Chemical synthesis method.
• 80% of lysine is produced by the fermentation process and 20% by chemical synthesis.
• To produce lysine number of micro-organisms are used like Yeasts, bacteria, and fungi.
• Industrial production of lysine is done by species like Corynebacterium glutamicum, Brevibacterium flavum, Brevibacterium lactofermentum, Corynebacterium Lilium, Brevibacterium divaricatum, and Escherichia coli.
• Industrial production of lysine can be carried out in two different ways, submerged fermentation and Aerobic Fermentation.
• Production of lysine is completed in three steps upstream, fermentation, and downstream process.
• Lysine is mostly produced from carbohydrates-based media using submerged fermentation, a variety of raw materials such as molasses-like cane and wheat are suitable for lysine production.
• The optimum temperature used for the production of lysine is between 30-40°C. 
• The optimum pH is about 7.2
• Various factors affect lysine production like carbon and nitrogen source, Temperature, pH, Aeration and Agitation, Nutrient Concentration, Trace elements, etc.
• Lysine has numerous applications in the Food industry. 

History of Lysine Production:

• Lysine was first isolated by the German biological chemist Ferdinand Heinrich Edmund Drechsel in 1889 from the protein casein in milk. He named it "lysin". 
• In 1902, the German chemists Emil Fischer and Fritz Weigert determined lysine's chemical structure by synthesizing it.
• In 1978, the first fermented L- lysin was produced by the Japanese company “kyowa Hokko Kogyo”. 
• The recent method of lysin production is based on the fermentation of carbohydrates.

Microorganisms used for Lysine Production:

• Different types of microorganisms have different abilities to produce Lysine. Therefore, different microorganisms can be used to produce Lysine products that people need. 
• Today, Lysine fermentation technology plays a huge role in the food, Pharma, and Medical industry. 
• To produce Lysine large number of micro-organisms are used like Yeasts, bacteria, and fungi. 
• Lysine is mainly produced by submerged fermentation using Corynebacterium glutamicum and Escherichia coli. from different sources of carbohydrates, such as molasses and sucrose-based media. 
• Other species like Brevibacterium flavum, Brevibacterium lactofermentum, Corynebacterium Lilium, and Brevibacterium divaricatum are used for lysine production.
• Homoserine auxotrophs strains or Genetically Engineered strains such as data (Dihydrodipicolinate Synthase), lysC (Aspartate Kinase), dpaB (Dihydrodipicolinate Reductase),  lysE (lysine export carrier gene) are used for lysine production.
• The main advantages of using this microorganism are: that it is easier to obtain and easy to handle its ease of handling. It gives maximum yield and can ferment a variety of cheap raw materials like molasses and starch.

Raw Materials used for Lysine Production: 

Although lysine is mostly produced from carbohydrates or sucrose-based media using liquid fermentation, a variety of raw materials such as molasses-like cane and wheat are suitable for lysine production.

Fermentation Media: 

1. Carbon Source: Cane Molasses
2. Nitrogen Source: Corn steep liquor / Soybean meal 
3. Minerals and Salts: KH2PO4/K2HPO4, CaCO3 
4. Trace Elements: Corn steep liquor
5. Antifoaming Agents: PEG-2000, Silicone-based oils 6. 

Stages of L-lysine Fermentation: 

Industrial fermentation of lysine requires three most important stages to complete product (lysine) fermentation. These are upstream processes, and actual fermentation and the last one is a downstream process.

1. Upstream process of fermentation:

• The upstream process includes the initial steps of a fermentation process.
• The upstream process of lysine fermentation comprises initial tasks of screening and identifying suitable microorganisms, and their improvement, Substrate selection, and Culture media preparation to enhance productivity and yield. 
• It also includes the maintenance of strain purity, preparation of inoculum, and Sterilization of media and bioreactor to improve the economic efficiency of the process.
• The seed Inoculum must contain Glucose, Biotin, Leucine, MgSO4, KH2PO4/K2HPO4, MnSO4, CaCO3 (NH4)2SO4.

2. Actual Fermentation Process:

• Fermentation is a process in which complex substances are converted into simple substances with the help of microorganisms.
• In this process, all the materials are added together in a bioreactor to produce the desired product.
• Maintaining optimal conditions required for lysine production lilk nutrients, are supplied for the growth and multiplication of microorganisms inside a bioreactor, Temp, pH, Oxygen concentration, etc.
• The fermentation is carried out at 30 to 40°C and is allowed for up to 100 hours. 
• The number of trace elements and growth factors like methionine, homoserine, and threonine should be appropriate for the maximum growth of microorganisms. 

3. Down-stream process:

• The downstream process of fermentation involves in the recovery of products from the bioreactor through different processes like extraction, separation, purification, and packaging of the product.
• Here the fermentation broth is sent to an ultrafiltration system for the removal of cell debris and other suspended solids. 
• Subsequently, the liquor from ultrafiltration is fed to ion-exchange columns, where L-lysine is selectively adsorbed. The adsorbed L-lysine is eluted from the ion-exchange resins by washing with an aqueous ammonia solution.
• Now the eluted L-lysine is mixed with mother liquor from the product filtration step and concentrated by evaporation. 
• The concentrated lysine solution is acidified with hydrochloric acid, and free L-lysine is converted to L- lysine HCl.
• The L-lysine HCl solution is then sent to the crystallizer, and lysine salt is filtered. The mother liquor is recycled to the evaporator and the wet cake is conveyed to dryers. 
• Final dry L-lysine-HCl (98.5 wt.%) is obtained and sent to a packaging line before being stored in bags.

The technique of L-lysine Production by Submerged Fermentation:

• Substrate use as Wheat molasses, Cane molasses, etc.
• The submerged fermentation method of L-Lysine production has easy control mechanisms and it lowers labor costs,
• This technique gives higher productivity and yield.
• The only disadvantage of this technique is it requires high media cost, is sensitive to trace metal inhibition, and has high wastewater generation.
• According to statistics, 80% of the L-lysine in the world comes from the liquid submerged fermentation of Corynebacterium glutamicum, and the medium is carbohydrates or raw materials containing glucose and sucrose.
• In addition, some agricultural processing wastes and their by-products can be used as carbon sources in the submerged fermentation of lysine.
• Compared with other fermentation types, submerged fermentation has significant advantages, such as high productivity and low labor costs.
• Both batch fermentation and fed-batch fermentation can be used in submerged fermentation, but batch fermentation is still the main method.

L-lysine Uses:

• L-lysine can promote calcium absorption and prevent osteoporosis. 
• Lysine is widely used in the fields of feed, food, medicine, and new chemical materials.
• Lysine can also be widely used in food additives, pharmaceutical infusions, and other fields.
• Lysine can promote human development, improve immunity, and enhance gastric juice secretion and bone marrow hematopoietic function. 
• Lysine plays an important role in regulating the metabolic balance in animals, improving the absorption of protein in the body, improving dietary nutrition, and promoting growth and development.
• Lysine is used in the cosmetic industry as a hair conditioner, As a beauty and anti-aging product.
• As an amino acid, lysine is an essential component of protein and an important component of carnitine production.
• Lysine can promote the healing of wound surfaces and cartilage damage.

Factors Affecting L-lysine Production:

1. Temperature : 

• The effect of temperature on L-lysine production is numerous. 
• The optimum temperature used for the production of L-lysine by submerged fermentation of Corynebacterium glutamicum and other bacteria is between 30 to 40°C.
• Temperature can affect the growth of Corynebacterium glutamicum activity. In the optimum temperature range, as the temperature increases the growth and metabolism of the Corynebacterium glutamicum accelerate, and the rate of the fermentation reaction increases. 
• When the temperature exceeds the optimal temperature range as the temperature rises the Corynebacterium glutamicum activity is quickly inactivated and the fermentation rate is reduced. 
• Therefore, to ensure the normal fermentation process, it is necessary to maintain the optimum temperature.

2. pH : 

• pH can affect the activity of microorganisms, enzymes, and fermentation rate. 
• Based on the type of substrate and media and microorganisms the pH is vary. 
• In defined media, the initial pH of the medium is usually adjusted to 7.2-7.3, while in the case of molasses the pH must be neutral or slightly acidic. 
• The pH of the medium is adjusted with NaOH, H2SO4, and HCl.  
• In addition, pH will also affect the decomposition of nutrients in the medium. Therefore, the pH of the fermentation broth should be controlled.

3. Dissolved oxygen Concentration : 

• The supply of oxygen is a key factor for aerobic fermentation and therefore it requires oxygen. 
• Aeration and agitation meet the oxygen demand of a fermentation process of L-lysine 
• Agitation is important for heat transfer, adequate mixing, and mass transfer.
• Aeration and agitation both maintain the homogeneous environment of culture by continuous mixing. 
• Therefore, a large amount of oxygen must be continuously added to the fermentation broth, and stirring can increase the solubility of oxygen in the fermentation broth. 
• The concentration of dissolved oxygen increased with the increase in the speed of agitation.

4. Concentration of nutrients : 

• The concentration of various nutrients in the fermentation broth, especially the ratio of carbon to nitrogen, inorganic salts, and vitamins, will directly affect the growth of bacteria and the production of L-lysine.