Fermentation Introduction

Introduction

Pharmaceutical proteins produced via fermentation in transgenic microbes or mammalian cell culture systems provide economical systems for production of therapeutic proteins. These include antibodies, vaccines, blood proteins, etc.

Biopharmaceuticals are medical drugs (see pharmacology) produced using biotechnology. They are proteins (including antibodies), nucleic acids (DNA, RNA or antisense oligonucleotides) used for therapeutic or in vivo diagnostic purposes, and are produced by means other than direct extraction from a native (non-engineered) biological source

Dozens of new pharmaceuticals produced via fermentation in transgenic microbes have been approved for therapeutic use in the USA. Hundreds of additional biotech drug candidates are in various stages of research or clinical trials. Fermentation systems can be scaled up to produce quantities of pharmaceuticals that are difficult or impossible to produce via traditional methods. Pharmaceutical quality may also be improved. For example, pharmaceuticals produced from blood must be carefully purified to ensure no transmission of viruses as accidental contaminants in the pharmaceutical product. Microbial systems that do not allow human viruses to replicate enable pharmaceutical production with little or no risk of virus contamination.

The total biotech exports in 2005-06 were at Rs 3,357.17 crore, while the domestic business reported Rs 3,163.83 crore in sales. The exports accounted for 51.48 percent share of the total industry. BioPharma exports accounted for 74.33 percent of the total exports of Rs 3,357.17 crore, clocking Rs 2,495.24 crore in revenues. BioAgri's share of exports was the lowest at 1.07 percent and exports from this segment stood at Rs 35.88 crore. BioServices overseas revenues were Rs 684 crore. BioIndustrial and Bioinformatics sector accounted for 1.23 percent and 3 percent share of the total exports respectively.

Classification of biopharmaceuticals

• Blood factors (Factor VIII and Factor IX)
• Thrombolytic agents (tissue plasminogen activator)
• Hormones (Insulin, growth hormone, gonadotrophins)
• Haematopoietic growth factors (Erythropoietin, colony stimulating factors)
• Interferons (Interferons-α, -β, -γ)
• Interleukin-based products (Interleukin-2)
• Vaccines (Hepatitis B surface antigen)
• Monoclonal antibodies (Various)
• Additional products (tumour necrosis factor, therapeutic enzymes)
• 
  

Definition of Fermentation:

Fermentation technology is the oldest of all biotechnological processes. The term is derived from the Latin verb fevere, to boil--the appearance of fruit extracts or malted grain acted upon by yeast, during the production of alcohol.

Fermentation is a process of chemical change caused by organisms or their products, usually producing effervescence and heat.

Microbiologists consider fermentation as 'any process for the production of a product by means of mass culture of micro-organisms'.

Biochemists consider fermentation as 'an energy-generating process in which organic compounds act both as electron donors and acceptors'; hence fermentation is ‘an anaerobic process where energy is produced without the participation of oxygen or other inorganic electron acceptors’.

One process by which carbon-containing compounds are broken down in an energy yielding process. Fermentation occurs during times of low oxygen supply and is therefore known as a type of anaerobic respiration.

The anaerobic enzymatic conversion of organic compounds, especially carbohydrates, to simplercompounds , especially to ethyl alcohol, resulting in energy in the form of adenosine Triphosphate (ATP).

The process is used in the production of alcohol, bread, vinegar and other food or industrial products. It differs from respiration in that organic substances rather than molecular oxygen are used as electron acceptors

Fermentation occurs widely in bacteria and yeasts, the process usually being identified by the product formed, for example, acetic, alcoholic, butyric and lactic fermentation are those that result in the formation of acetic acid, alcohol, butyric acid and lactic acid, respectively.


Commercially important Fermentation

1. Microbial cells or Biomass as the product: Eg. Bakers Yeast, Lactic acid bacillus, Bacillus sp.
2. Microbial Enzymes: Catalase, Amylase, Protease, Pectinase, Glucose isomerase, Cellulase, Hemicellulase, Lipase, Lactase, Streptokinase etc.
3. Microbial metabolites :
   1. Primary metabolites – Ethanol, Citric acid, Glutamic acid, Lysine, Vitamins, Polysaccharides etc.
   2. Secondary metabolites: All antibiotic fermentation
4. Recombinant products : Insulin, HBV, Interferon, GCSF, Streptokinase
5. Biotransformations: Eg. Phenyl acetyl carbinol,Steroid Biotransformation

Nutrient sources for industrial fermentation

Medium for Industrial Fermentations

Any Microbe requires Water, Oxygen, Energy source, Carbon source, Nitrogen source and Micronutrients for the growth.

Carbon & Energy source + Nitrogen source + O₂ + other requirements → Biomass + Product + byproducts + CO₂ + H₂O + heat

Nutrient	Raw material
Carbon
Glucose	Corn sugar, Starch, Cellulose
Sucrose	Sugarcane, Sugar beet molasses
Lactose	Milk whey
Fats	Vegetable oils
Hydrocarbons	Petroleum fractions
Nitrogen
Protein	Soybean meal, Cornsteep liquor, Distillers' solubles
Ammonia	Pure ammonia or ammonium salts Urea
Nitrate	Nitrate salts
Phosphorus source	Phosphate salts

Nutrient sources for industrial fermentation

Nutrient Raw material

Carbon source

Glucose Corn sugar, Starch, Cellulose

Sucrose Sugarcane, Sugar beet molasses

Lactose Milk whey

Fats Vegetable oils

Hydrocarbons Petroleum fractions

Nitrogen source

Protein Soybean meal, Cornsteep liquor, Distillers' solubles

Ammonia Pure ammonia or ammonium salts

Nitrate Nitrate salts

Nitrogen Air

Phosphorous source Phosphate salts

Trace elements : Fe, Zn, Cu, Mn, Mo, Co

Antifoaming agents : Esters, Fatty acids, Silicones, Sulphonates, Polypropylene

Buffers: Calcium carbonate, Phosphates

Growth factors: Some microorganisms cannot synthesize the required cell components themselves and need to be supplemented: E.g. Thiamine, Biotin, Calcium pentothenate

Precursors: Directly incorporated into the desired product: Phenyl ethylamine into Benzyl penicillin, Phenyl acetic acid into Penicillin G

Inhibitors: To get the specific products: e.g. Sodium barbital for Rifamycin

Inducers: Majority of the enzymes are inducible and are synthesized in response of inducers: e.g. Starch for Amylases, Maltose for Pollulanase, Pectin for Pectinase

Chelators: Chelaters are the chemicals used to avoid the precipitation of metal ions. Chelaters like EDTA, Citric acid, Polyphosphates are used in low concentrations.

For more details on industrial fermentation read

1. Biochemical Engineering Fundamentals by J.E. Bailey and P.F. Ollis, McGraw Hill Publication 2. . Principles of fermentation technology by Stansbury, P.F., A. Whitaker and S.J. Hall, 1997

Biotechnology Companies Developing Pharmaceuticals via Fermentation in Transgenic Microbes or Mammalian Cell Cultures

• Atlantic BioPharmaceuticals, Inc.
• Chiron Corporation
• BioPharmaceuticals Pipeline
• Genentech, Inc.
• Interomex Biopharmaceuticals
• Lonza Biotechnology
• Marathon Biopharmaceuticals, Inc.
• NPS Pharmaceuticals
• Protein Design Labs
• Protein Sciences Corp

PRODUCTS OF FERMENTATION PROCESSES

The growth of micro-organisms or other cells results in a wide range of products. Each culture operation has one or few set objectives. The process has to be monitored carefully and continuously, to maintain the precise conditions needed and recover optimum levels of products. Accordingly, fermentation processes aim at one or more of the following:

a) production of cells (biomass) such as yeasts;

b) extraction of metabolic products such amino acids, proteins (including enzymes), vitamins, alcohol, etc., for human and/or animal consumption or industrial use such as fertiliser production;

c) modification of compounds (through the mediation of elicitors or through biotransformation); and

d) production of recombinant products.

A. MICROBIAL BIOMASS

Microbial biomass is produced commercially as single cell protein (SCP) using such unicellular algae as species of Chlorella or Spirulina for human or animal consumption, or viable yeast cells needed for the baking industry, which was also used as human feed at one time. Bacterial biomass is used as animal feed. The biomass of Fusarium graminearum is also produced, for a similar use.

B. MICROBIAL METABOLITES

i) Primary metabolites:

During the log or exponential phase organisms produce a variety of substances that are essential for their growth, such as nucleotides, nucleic acids, amino acids, proteins, carbohydrates, lipids, etc., or by- products of energy yielding metabolism such as ethanol, acetone, butanol, etc. This phase is described as the tropophase, and the products are usually called primary metabolites. Commercial examples of such products are given in Table 2.

TABLE 2

Examples of commercially produced primary metabolites

Primary Organism Significance

Metabolite

Ethanol Saccharomyces cerevisiae alcoholic beverages

Kluyveromyces fragilis

Citric acid Aspergillus niger food industry

Acetone and Clostridium

butanol acetobutyricum solvents

Lysine Corynebacterium nutritional additive

Glutamic acid glutamacium flavour enhancer

Riboflavin Ashbya gossipii nutritional

Eremothecium ashbyi

Vitamin B12 Pseudomonas denitrificans nutritional

Propionibacterium shermanii

Dextran Leuconostoc mesenteroides industrial

Xanthan gum Xanthomonas campestris industrial

ii) Secondary metabolites:

Organisms produce a number of products, other than the primary metabolites. The phase, during which products that have no obvious role in metabolism of the culture organisms are produced, is called the idiophase, and the products are called secondary metabolites.

In reality, the distinction between the primary and secondary metabolites is not a straightjacket situation. Many secondary metabolites are produced from intermediates and end products of secondary metabolism. Some like those of the Enterobacteriaceae do not undergo secondary metabolism. Examples of secondary metabolites are given in Table 3.

TABLE 3

Examples of commercially produced secondary metabolites

Metabolite Species Significance

Penicillin Penicillium chrysogenum antibiotic

Erythromycin Streptomyces erythreus antibiotic

Streptomycin Streptomyces griseus antibiotic

Cephalosporin Cephalosporium acrimonium antibiotic

Griseofulvin Penicillium griseofulvin antifungal antibiotic

Cyclosporin A Tolypocladium inflatum immunosuppressant

Gibberellin Gibberella fujikuroi plant growth regulator

Secondary metabolism may be repressed in certain cases. Glucose represses the production of actinomycin, penicillin, neomycin and streptomycin; phosphate represses streptomycin and tetracyclin production. Hence, the culture medium for secondary metabolite production should be carefully chosen.

C. PRODUCTION ENZYMES

Industrial production of enzymes is needed for the commercial production of food and beverages. Enzymes are also used in clinical or industrial analysis and now they are even added to washing powders (cellulase, protease, lipase). Enzymes may be produced by microbial, plant or animal cultures. Even plant and animal enzymes can be produced by microbial fermentation. While most enzymes are produced in the tropophase, some like the amylases (by Bacillus stearothermophilus) are produced in the idiophase, and hence are secondary metabolites. Examples of enzymes produced through fermentation processes are given in Table 4.

TABLE 4

Examples of commercially produced enzymes

Organism Enzyme

Aspergillus oryzae Amylases

Aspergillus niger Glucamylase

Trichoderma reesii Cellulase

Saccharomyces cerevisiea Invertase

Kluyveromyces fragilis Lactase

Saccharomycopsis lipolytica Lipase

Aspergillus species Pectinases and proteases

Bacillus species Proteases

Mucor pusillus Microbial rennet

Mucor meihei Microbial rennet

D. FOOD INDUSTRY PRODUCTS

A very wide range of innumerable products of the food industry, such as sour cream, yoghurt, cheeses, fermented meats, bread and other bakery products, alcoholic beverages, vinegar, fermented vegetables and pickles, etc., are produced through microbial fermentation processes. The efficiency of the strains of the organisms used, and the processes are being continuously improved to market quality products at more reasonable costs.

E. RECOMBINANT PRODUCTS

Recombinant DNA technology has made it possible to introduce genes from any organism into micro-organisms and vice versa, resulting in transgenic organisms and the latter are made to produce the gene product. Genetically manipulated Escherichia coli, Saccharomyces cerevisiae, other yeasts and even filamentous fungi are now being used to produce interferon, insulin, human serum albumin, and several other products.

F. BIOTRANSFORMATION

Production of a structurally similar compound from a particular one, during the fermentation process is transformation, or biotransformation, or bioconversion. The oldest instance of this process is the production of acetic acid from ethanol.

Immobilised plant cells may be used for biotransformation. Using alginate as the immobilising polymer, digitoxin from Digitalis lanata was converted into digoxin, which is a therapeutic agent in great demand. Similarly, codeinone was converted into codeine and tyrosine from Mucuna pruriens was converted into DOPA.

G. ELICITORS

It is possible to induce production or enhance production of a compound in cultures by using elicitors, which may be micro-organisms. For example, Saccharomyces cerevisiae was an efficient elicitor in the production of glyceollin (Glycine max) and berberine (Thalictrum rugosum). Rhizopus arrhizus trebled diosgenin production by Dioscorea deltoidea. The production of morphine and codeine by Papaver somniferum was increased 18 times by Verticillium dahliae.

EXAMPLES :

Vitamins

• Figure 11.13, Vitamin B₁₂
  
• Used as supplements for human food and animal feeds
  
• Nearly $1B/year
  
• Synthesized chemically, but some by biocatalysis
  
• Selected high-yield strains for B₁₂ produce up to 60 mg/L
  
• For riboflavin, up to 7 g/L
  

Amino Acids in the Food Industry (a short list)

• L-Glutamate (MSG) flavor enhancer
  
• Aspartame (phe + asp) sweetener
  
• L-Lysine nutritive additive
  
• DL- Methionine nutritive additive
  

Industrial Production of Lysine

• Figure 11.14a, Biochemical pathway of aspartate to lysine
  
• Figure 11.14g, Structures of lysine and S-aminoethylcysteine
  
• Overproducing strain: Brevibacterium flavum can produce over 60 g lysine/liter

Bioconversion

• Supplement to organic synthesis
  
• Growth in a large fermentor
  
• Chemical to be converted added at appropriate time
  
• Incubation
  
• Conversion
  
• Extraction
  
• Purification of product
  
• Figure 11.15, Cortisone production using a microorganism
  

Enzymes and their Markets

• Pharmaceuticals
  
  • Chiral resolution ($25B)
    
• Agricultural
  
  • Genetically engineered crops, animal feed ($5.9B)
    
• Chemical
  
  • Fine chemicals ($45B), specialty chemicals, and polymers
    
• Industrial
  
  • Consumer products, oil well breakers
    

Enzymes and Industrial Uses

• Amylases = used in bread, glucose manufacture, in detergents
  
• Proteases = used in bread, stain removal, meat tenderizing, contact cleaning solns
  
• Cellulases = used in fabric softening, “stone-washed” jeans, detergents
  
• Lipases = used in detergents, break-down of fats
  
• DNA polymerases = used in biotechnology (PCR)
  

“Extremo”zymes: Enzymes that function under harsh industrial and environmental conditions

• There is a need for enyzmes that catalyze desired (and often very specific) chemical conversions under typically harsh industrial and environmental conditions:
  
  • Low- and High pH
    
  • Cold to very hot temperatures
    
  • Activity in non-aqueous solutions
    

Thermostable Enzymes

• Figure 11.16b, Thermostability of pullulanase from Pyrococcus woesei
  
• Examples of “Extremo”zymes
  
• Thermus aquaticus
  
  • DNA polymerase @ 75 – 95 C
    
• Thermus thermophilus
  
  • Pyrophosphatase @ 75 C
    
• Thermococcus litoralis
  
  • Pullulanase @ 118 C
    
• Pyrococcus furiosus
  
  • Ferredoxin @ 140 C
    
• Psychrophiles – cold active enzymes
  
• Acidophiles/Alkaliphiles – low-/high pH
  
• Halophiles – High salt concentrations