Name ; Reema Amin

Registration no: FA19-BSO-058

Topic: Fermentation processes (primary and secondary metabolites), downstream
processing/ product recovery methods, purification

Fermentation:
Fermentation is the process in which microorganisms are used to break a substance into simpler
substances. Fermentation is an enzyme catalyzed, metabolic process whereby organisms convert starch or
sugar to alcohol or an acid anaerobically releasing energy. The science of fermentation is called
“zymology”.
Microorganisms like yeast and bacteria usually play a role in fermentation process, creating beer, wine,
bread, kimchi, yogurt and other foods.
Types of fermentation
Homo fermentation: only one type of product formation
Hetero fermentation: more than one product formed
On the bases of the end product formation, it has the following types

1-Lactic acid fermentation

Lactic acid is formed from pyruvate produced in glycolysis. NAD+ is generated from NADH.
Enzyme lactate dehydrogenase catalyzes this reaction. Lactobacillus bacteria prepare curd from
milk via this type of fermentation. During intense exercise when oxygen supply is inadequate,
muscles derive energy by producing lactic acid, which gets accumulated in the cells causing
fatigue.

2-Alcoholic fermentation
This is used in the industrial production of wine, beer, biofuel, etc. The end product is alcohol
and CO2. Pyruvic acid breaks down into acetaldehyde and CO2 is released. In the next step,
ethanol is formed from acetaldehyde. NAD+ is also formed from NADH, utilized in glycolysis.
Yeast and some bacteria carry out this type of fermentation. Enzyme pyruvic acid decarboxylase
and alcohol dehydrogenase catalyse these reactions.

3-Acetic acid fermentation

Vinegar is produced by this process. This is a two-step process.
The first step is the formation of ethyl alcohol from sugar anaerobically using yeast.

In the second step, ethyl alcohol is further oxidized to form acetic acid using acetobacter
bacteria. Microbial oxidation of alcohol to acid is an aerobic process.

Butyric acid fermentation

This type of fermentation is characteristic of obligate anaerobic bacteria of genus clostridium. This
occurs in retting of jute fiber, rancid butter, tobacco processing and tanning of leather. Butyric acid is
produced in the human colon as a product of dietary fiber fermentation. It is an important source of
energy for colorectal epithelium. Sugar is first oxidized to pyruvate by the process of glycolysis and then
pyruvate is further oxidized to form acetyl-CoA by the oxidoreductase enzyme system with the
production of H2 and CO2. Acetyl-CoA is further reduced to form butyric acid. This type of fermentation
leads to a relatively higher yield of energy. 3 molecules of ATP are formed.

Primary and secondary metabolites

1-Primary and secondary metabolites are used in fermentation process for the production of food,
amino acids and antibiotics. Primary metabolites are considered essential to microorganisms for proper
growth A primary metabolite is typically present in many organisms or cells. It is also referred to as a
central metabolite, which has an even more restricted meaning (present in any autonomously growing
cell or organism). Some common examples of primary metabolites include: ethanol, lactic acid, and
certain amino acids..
2-Secondary metabolites do not play a role in growth, development, and reproduction, and are formed
during the end or near the stationary phase of growth. They are organic compounds which are not
directly involved in survival of plants but they produce some produces which aid them in their normal
growth and development. Secondary metabolites are compounds biosynthetically derived from primary
metabolites but more limited in distribution in the plant kingdom, being restricted to a particular
taxonomic group (species, genus, family, or closely related group of families).

These metabolites can be used in industrial microbiology to obtain amino acids, develop
vaccines and antibiotics, and isolate chemicals necessary for organic synthesis.

Downstream processing:
Downstream processing is the series of operations required to take biological material such as
cells , tissue culture fluid, or plant tissues, derive from them a pure and homogeneous protein
product.
  The selection of suitable process of recovery and purification depends upon the nature of
the end product, their concentration, the by-products present, the stability of the product
and degree of purification.

Depending on the nature of the product and method of synthesis, Downstream Processing (DSP)
generally includes a combination of the following steps:

 Harvest and Filtration

Harvest is the first step to separate product from bulk debris while optimizing retention of the
product yield and quality
 Primary Capture
Capture refers to holding onto the desired product while minimizing retention of impurities and
by-products. It differs from 'Harvest' in that essentially all bulk debris will have already been
removed.

 Buffer Exchange and Up-concentration

Ultrafiltration (UF) concentrates a dilute product stream and separates molecules in solution
based on the membrane pore size or molecular weight cutoff. Diafiltration (DF) exchanges
products from an existing buffer into a new buffer for a following process or a final formulation
buffer. Ultrafiltration (UF) and diafiltration (DF) together are the steps of buffer exchange.

 Purification (and Contaminant or Impurity Clearance)

Purification removes residual impurities with each successive method employed, while also
retaining as much product as possible. The goal is to achieve product purity while minimizing
potential yield loss.

 Bioconjugation (Molecule Dependent)

Bioconjugate molecules are a new class or generation of biologic molecules which are designed
to have an increased efficacy enabled by the combined function of two or more different
therapeutic types of molecules. Antibody-Drug Conjugates (ADC) are one of the more common
bio conjugates and are synthesized by biochemically modifying an antibody and covalently
linking it to another active pharmaceutical ingredient (API).

 Formulation
Formulation is the process that transitions a drug substance (DS) into a formulated drug product
(DP). Formulation brings the product molecule from an environment, solvent or other physical
state into a form suitable for clinical administration.

Product Recovery methods

Product recovery is another alternative, where applied potential is used to produce various value
added products through microbial electrolysis cell (MEC) and microbial electrosynthesis (MES)
process

Various techniques involved in product recovery are filtration, centrifugation, precipitation,

dialysis, solvent extraction, chromatographic and electrophoretic techniques, crystallization
techniques etc. As we get our resultant filtrate, then this filtrate is subjected for the rest of the
purification process.

Either we use product recovery method or down streaming process after fermentation has been
done.

Purification :
Purification in a chemical context is the physical separation of a chemical substance of interest
from foreign or contaminating substances. Pure results of a successful purification process are
termed isolate.

purification by Chromatography:
The biological products of fermentation (proteins, pharmaceuticals, diagnostic compounds and
research materials) are very effectively purified by chromatography. It is basically an analytical
technique dealing with the separation of closely related compounds from a mixture. Chromato-
graphy usually consists of a stationary phase and mobile phase.
Membrane Filtration :
A membrane is a thin layer of semi-permeable material that separates substances when a driving
force is applied across the membrane. Membrane processes are increasingly used for removal of
bacteria, microorganisms, particulates, and natural organic material, which can impart color,
tastes, and odors to water and react with disinfectants to form disinfection byproducts. As
advancements are made in membrane production and module design, capital and operating costs
continue to decline. The membrane processes discussed here are microfiltration (MF),
ultrafiltration (UF), Nano filtration (NF), and reverse osmosis (RO).
Precipitation:
Precipitation is the most commonly used technique in industry for the concentration of
macromolecules such as proteins and polysaccharides. Further, precipitation technique can also
be employed for the removal of certain unwanted byproducts e.g. nucleic acids, pigments.

Neutral salts, organic solvents, high molecular weight polymers (ionic or non-ionic), besides
alteration in temperature and pH are used in precipitation. In addition to these non-specific
protein precipitation reactions (i.e. the nature of the protein is unimportant), there are some
protein specific precipitations e.g., affinity precipitation, ligand precipitation.

https://courses.lumenlearning.com/wm-biology1/chapter/reading-types-of-fermentation/

https://byjus.com/neet/types-of-fermentation/

http://www.jnkvv.org/PDF/11042020204520primary%20and%20secondary%20metabolites%20and%20
their%20applications%20(3%20files%20merged).pdf

https://www.sciencedirect.com/topics/engineering/downstream-processing

https://www.mt.com/sg/en/home/applications/L1_AutoChem_Applications/fermentation/downstream
-processing-in-biotechnology.html
https://www.biologydiscussion.com/biotechnology/downstream-processing/stages-in-downstream-
processing-5-stages/10160