Fermentation

Fermentation is a metabolic process that converts sugar to acids, gases or alcohol. It occurs in
yeast and bacteria, and also in oxygen-starved muscle cells, as in the case of lactic acid
fermentation. Fermentation is also used more broadly to refer to the bulk growth of
microorganisms on a growth medium, often with the goal of producing a specific chemical
product like enzyme, vaccines, antibiotics, food product/additive etc. French microbiologist
Louis Pasteur is often remembered for his insights into fermentation and its microbial causes. The
science of fermentation is known as zymology.

Fermentation takes place in the lack of oxygen (when the electron transport chain is unusable) and
becomes the cell’s primary means of ATP (energy) production. It turns NADH and pyruvate
produced in the glycolysis step into NAD+ and various small molecules depending on the type of
fermentation. In the presence of O2, NADH and pyruvate are used to generate ATP in respiration.
This is called oxidative phosphorylation, and it generates much more ATP than glycolysis alone.
For that reason, cells generally benefit from avoiding fermentation when oxygen is available, the
exception being obligate anaerobes which cannot tolerate oxygen.

The first step, glycolysis, is common to all fermentation pathways:

C6H12O6 + 2 NAD+ + 2 ADP + 2 Pi → 2 CH3COCOO− + 2 NADH + 2 ATP + 2 H2O +

2H+

Pyruvate is CH3COCOO−. Pi is inorganic phosphate. Two ADP molecules and two Pi areconverted
to two ATP and two water molecules via substrate-level phosphorylation. Two molecules of NAD+
are also reduced to NADH. In oxidative phosphorylation the energy for ATP formation is derived
from an electrochemical proton gradient generated across the inner mitochondrial membrane (or,
in the case of bacteria, the plasma membrane) via the electron transport chain. Glycolysis has
substrate-level phosphorylation (ATP generated directly at the point of reaction).

Humans have used fermentation to produce food and beverages since the Neolithic age. For
example, fermentation is used for preservation in a process that produces lactic acid as found in
such sour foods as pickled cucumbers, kimchi and yogurt, as well as for producing alcoholic
 beverages such as wine and beer. Fermentation can even occur within the stomachs of animals,
such as humans.

Definitions of Fermentation

Fermentation is an anaerobic biochemical process. In the process of fermentation, the

first step is the same as cellular respiration, which is the formation of pyruvic acid by
glycolysis where net 2 ATP molecules are synthesised. In the next step, pyruvate is
reduced to lactic acid, ethanol or other products. Here NAD+ is formed which is re-
utilized back in the glycolysis process.

Examples of Fermentation

Fermentation does not necessarily have to be carried out in an anaerobic environment. For
example, even in the presence of abundant oxygen, yeast cells greatly prefer fermentation to
aerobic respiration, as long as sugars are readily available for consumption (a phenomenon known
as the Crabtree effect). The antibiotic activity of hops also inhibits aerobic metabolism in yeast.
Fermentation react NADH with an endogenous, organic electron acceptor. Usually this is pyruvate
formed from the sugar during the glycolysis step. During fermentation, pyruvate is metabolized to
various compounds through several processes:

1. Ethanol fermentation, aka alcoholic fermentation, is the production of ethanol and

 carbon dioxide
2. Lactic acid fermentation refers to two means of producing lactic acid:

• Homolactic fermentation is the production of lactic acid exclusively

Heterolactic fermentation is the production of lactic acid as well as other acids and alcohols.
Lactic Acid Fermentation
Lactic acid is formed from pyruvate produced in glycolysis. NAD+ is generated from NADH.
Enzyme lactate dehydrogenase catalyses this reaction. Lactobacillus bacteria prepare curd from
milk by 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

Alcohol 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 which is reused in glycolysis. Yeast
and some bacteria carry out this type of fermentation. Enzyme pyruvic acid decarboxylase and
alcohol dehydrogenase catalyse these reactions.

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 oxidised 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 fibre, rancid butter, tobacco processing and tanning of leather. Butyric acid
is produced in the human colon as a product of dietary fibre fermentation. It is an important source
of energy for colorectal epithelium. Sugar is first oxidised to pyruvate by the process of glycolysis
and then pyruvate is further oxidised to form acetyl-CoA by the oxidoreductase enzyme system
with the production of CO2 and H2. 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.
Hydrogen gas production in fermentation
Hydrogen gas is produced in many types of fermentation (mixed acid fermentation, butyric acid
fermentation, caproate fermentation, butanol fermentation, glyoxylate fermentation), as a way to
regenerate NAD+ from NADH. Electrons are transferred to ferredoxin, which in turn is oxidized by
hydrogenase, producing H2. Hydrogen gas is a substrate for methanogens and sulfate reducers,
which keep the concentration of hydrogen low and favor the production of such an energy-rich
compound, but hydrogen gas at a fairly high concentration can nevertheless be formed, as in flatus.

As an example of mixed acid fermentation, bacteria such as Clostridium pasteurianum ferment

glucose producing butyrate, acetate, carbon dioxide and hydrogen gas: The reaction leading to
acetate is:

C6H12O6 + 4 H2O → 2 CH3COO− + 2 HCO3− + 4 H+ + 4 H2

Glucose could theoretically be converted into just CO2 and H2, but the global reaction releases little
energy.
Methane gas production in fermentation
Acetic acid can also undergo a dismutation reaction to produce methane and carbon dioxide:
CH3COO− + H+ → CH4 + CO2 ΔG° = -36 kJ/reaction
This disproportionation reaction is catalysed by methanogen archaea in their fermentative
metabolism. One electron is transferred from the carbonyl function (e− donor) of the carboxylic
group to the methyl group (e− acceptor) of acetic acid to respectively produce CO2 and methane gas
ADVANTAGES OF FERMENTATION:

Fermentation is suitable for all kinds of environments. It is one of the oldest metabolic processes which is common to
prokaryotes and eukaryotes. Fermentation is widely used in various industries.
Using suitable microorganisms and specified conditions different kinds of products are formed namely:-

• Wine
• Beer
• Biofuels
• Yogurt
• Pickles
• Bread
• Sour foods containing lactic acid
• Certain antibiotics and vitamins
Production of biomass

Microbial cells or biomass is sometimes the intended product of fermentation. Examples include single cell protein,
baker’s yeast, Lactobacillus, E. coli, and others. In the case of single-cell protein, algae are grown in large open ponds
which allow photosynthesis to occur. If the biomass is to be used for inoculation of other fermentations, care must be
taken to prevent mutations from occurring.
Production of extracellular metabolites
Microbial metabolites can be divided into two groups: those produced during the growth phase of the organism, called
primary metabolites and those produced during the stationary phase, called secondary metabolites. Some examples of
primary metabolites are ethanol, citric acid, glutamic acid, lysine, vitamins and polysaccharides. Some examples of
secondary metabolites are penicillin, cyclosporin A, gibberellin, and lovastatin.
Primary metabolites
Primary metabolites are compounds made during the ordinary metabolism of the organism during the growth phase. A
common example is ethanol or lactic acid, produced during glycolysis. Citric acid is produced by some strains of
Aspergillus niger as part of the citric acid cycle to acidify their environment and prevent competitors from taking over.
Glutamate is produced by some Micrococcus species, and some Corynebacterium species produce lysine, threonine,
tryptophan and other amino acids. All of these compounds are produced during the normal "business" of the cell and
released into the environment. There is therefore no need to rupture the cells for product recovery.
Secondary metabolites
Secondary metabolites are compounds made in the stationary phase; penicillin, for instance, prevents the growth of
bacteria which could compete with Penicillium molds for resources. Some bacteria, such as Lactobacillus species, are
able to produce bacteriocins which prevent the growth of bacterial competitors as well. These compounds are of obvious
value to humans wishing to prevent the growth of bacteria, either as antibiotics or as antiseptics (such as gramicidin S).
Fungicides, such as griseofulvin are also produced as secondary metabolites. Typically secondary metabolites are not
produced in the presence of glucose or other carbon sources which would encourage growth, and like primary
metabolites are released into the surrounding medium without rupture of the cell membrane.
Production of intracellular components
Of primary interest among the intracellular components are microbial enzymes: catalase, amylase, protease, pectinase,
glucose isomerase, cellulase, hemicellulase, lipase, lactase, streptokinase and many others. Recombinant proteins, such
as insulin, hepatitis B vaccine, interferon, granulocyte colony-stimulating factor, streptokinase and others are also made
this way. The largest difference between this process and the others is that the cells must be ruptured (lysed) at the end
of fermentation, and the environment must be manipulated to maximize the amount of the product. Furthermore, the
product (typically a protein) must be separated from all of the other cellular proteins in the lysate to be purified.
Transformation of substrate
Substrate transformation involves the transformation of a specific compound into another, such as in the case of
phenylacetylcarbinol, and steroid biotransformation, or the transformation of a raw material into a finished product, in
the case of food fermentations and sewage treatment.
Food fermentation
Ancient fermented food processes, such as making bread, wine, cheese, curds, idli, dosa, etc., can be dated to more than
seven thousand years ago. They were developed long before man had any knowledge of the existence of the
microorganisms involved. Some foods such as Marmite are the byproduct of the fermentation process, in this case in the
production of beer.
Ethanol fuel
Fermentation is the main source of ethanol in the production of Ethanol fuel. Common crops such as sugar cane, potato,
cassava and corn are fermented by yeast to produce ethanol which is further processed to become fuel.
Sewage treatment
In the process of sewage treatment, sewage is digested by enzymes secreted by bacteria. Solid organic matters are
broken down into harmless, soluble substances and carbon dioxide. Liquids that result are disinfected to remove
pathogens before being discharged into rivers or the sea or can be used as liquid fertilizers. Digested solids, known also
as sludge, is dried and used as fertilizer. Gaseous byproducts such as methane can be utilized as biogas to fuel electrical
generators. One advantage of bacterial digestion is that it reduces the bulk and odor of sewage, thus reducing space
needed for dumping. The main disadvantage of bacterial digestion in sewage disposal is that it is a very slow process.
Agricultural Feed
A wide variety of agroindustrial waste products can be fermented to use as food for animals, especially ruminants. Fungi
have been employed to break down cellulosic wastes to increase protein content and improve in vitro digestibility.

ETHANOL PRODUCTION

Introduction
• Biological process in which sugars (glucose, fructose, sucrose) areconverted into
cellular energy.
• Ethanol is produced in the result of this process.
• It is colorless, volatile or flammable liquid.
 • It is widely used as biofuel as well as an alcoholic beverage isincreasingly being
consumed globally.
• This is mainly because of the cheap raw materials available.
Raw material
• Ethanol can be derived from either sugar, starchy materials or
lignocelluloses.
• The main feedstock for ethanol production includes sugarcane, sugarbeet, corn,
wheat.
Sugars:
• Sugarcane (molasses & juice)
• Cane sugar (clarified concentrated syrup)
• Sugar beet
Starchy materials:
• Corn
• Wheat
• Sweet sorghum
• Cassava
Lignocellulosic material:
• Sugarcane bagasse
• Corn stover
• Cereal straws
Feedstock conditioning and Pretreatment
Dilution:
• Molasses must be diluted to below to 25 °Bx (Brix) as yeast start toferment
quickly at this concentration.
Sedimentation:
• To prevent any incrustation in the pipelines or distillation towers due toash content
in molasses greater than 10%.
• The special chelating agent can also be used to remove the incrustation.
Addition of org & inorganic compounds:
• Done to offset the negative effect of salt which in turn increases theosmotic
pressure.
• Yeast strains resistant to salts are also developed.
Microfiltration:
• To remove the impurities that stick to the surface of the biocatalyst whenimmobilize
cells are used.
Microorganism involved
Bacteria used:
• Zymomonas mobilis
• Clostridium acetobutylicum
• E.coli
Yeast used:
• Saccharomyces cerevisiae
• Saccharomyces uvarum
• Candida utilis
• Kluyveromyces fragilis
Features of Microbes:
• Due to the small size, having a high surface ratio.
• Due to having a resistant cell wall, producing high concentrationsubstances leads
to a faster fermentation rate.
• The intense metabolism permits the development of a continuousfermentation
process.
 • Cells growth rate offsets at which cells are removed from the bioreactor.
• Have the ability to “predigest” the available food source and release bothproducts
and the intermediate metabolites.
• Using immobilized cells of microbes by treating with Calcium alginate toadsorbed on
the surface of materials.
• Using genetically modified microbes to enhance the fermentation
process.
Physical requirements:
• The ideal pH is around 4.0-4.5.
• The initial temperature is kept between 21-26 ºC.
• Ethanol gets evaporated at 27 ºC.
• Aeration is initially required for the growth of microbes.
• Later, anaerobic condition are created by withdrawing oxygen coupledwith the
production of carbon dioxide.
Chemical requirements

Nitrogen source:
• Urea is the most suitable source.
• Gaseous ammonium increases the pH of the medium.
• Ammonium sulfate can lead to incrustation.
Phosphorus source:
• Diammonium phosphate used as a source.
Hydrolytic enzymes:
• They can also be added to convert biopolymers and non-fermentablesubstances
in the molasses to monosaccharides or amino acids.
Process flow:
Classical fermentation can be achieved in three steps:
• During the first phase (22-24 h), yeast cells multiply aerobically byconsuming
oxygen present in the mash.
• In the middle phase (24-48 h), alcohol production occurs with postsaccharification of
sugars and multiplication of yeast falls off.
• The decrease in alcohol formation along with insignificant yeast growthat the final
stage (48-72 h).
Image Source: https://doi.org/10.1016/j.biortech.2018.02.125
Production process:
There are following steps in ethanol production:
• Milling
• Liquefaction
• Saccharification
• Fermentation
• Distillation
• Dehydration
1. Milling:
• The feedstock is passed through a hammer mill which grinds it into a finepowder
called a meal.
2. Liquefaction:
• The meal is mixed with water and alpha-amylase.
• Then passed through cookers where the starch is liquified and heat isapplied
here to enable liquefaction.
• Cookers with the high-temperature stage (120-150˚C) and lower
temperature holding period (95˚C) are used.
• High temperatures reduce bacteria levels in the mash.
3. Saccharification:
• The mash from the cookers is cooled.
 • And secondary enzyme glucoamylase is added.
• This converts the liquified starch into the fermentable sugars.
4. Fermentation:
• Yeast is added to ferment the sugars to ethanol and carbon dioxide.
• In a continuous process, the fermenting mash can flow through severalfermenters
until it is fully fermented and leaves the final tank.
• In a batch process, the mash stays in one fermenter for about 48 hoursbefore
distillation starts.
Batch fermentation
´Yeast reuse results in a decrease in new growth with no more sugar availablefor ethanol
production and an increase in the yield from 2 to 7%.
´Traditional yield 1-3g/L.
Continuous fermentation:
• To ensure system homogeneity and reduce the concentration gradient inthe culture
broth, CSTRs are employed.
• Reduce construction costs of bioreactors
• Lower requirements of maintenance and operation
• Better control of the process
• Higher productivities
• Cultivation of yeast under anaerobic conditions for a long time diminishtheir ability
to produce ethanol.
• Aeration is important which can enhance cell concentration.
Extraction of the final product:
5. Distillation:
• Mash is pumped to continuous flow.
• Multicolumn distillation system where the alcohol is removed from solidand water.
• The alcohol leaves the top of the final column at about 96% strength.
• The residue mash is called stillage which is transferred from the base ofthe column
to the co-product processing area.
6. Dehydration:
• The alcohol from the top of the column is passed through a dehydrationsystem
where the remaining water will be removed.
• Most ethanol plants use a molecular sieve to capture the last bit of waterin the
ethanol.
• The alcohol product at this stage is called anhydrous alcohol.
Fermentation Byproduct

Dried distiller grains with soluble (DDGS):

• The form is available to the feed industry.
• The liquid is separated from mash during the distillation process.
• It is partially dehydrated into syrup.
• Then added back on to the dried distiller’s grain to create DDGS.
Carbon dioxide:
• Used to carbonate the beverages.
• Manufacture dry ice.
• Used to flash freeze meat.
• Used by paper mills and food industries.

PENICILLIN PRODUCTION COMMERCIALLY BY FERMENTATIONBIOTECHNOLOGY

Structure of Penicillin:
▪ The basic structure of penicillin consists of a thiozolidine ring condensed with a B-
lactum ring.
▪ Natural penicillin is 6-amino-penicillinic acid (6APA).

Fermentation biotechnology for penicillin production:

▪ By fermentation technology penicillin is produced from Penicillium spp. If penicillin
fermentation is carried out without addition of side chain precursor, the natural
penicillins are produced. But fermentation can be better controlled by adding a side chain
precursor to obtain derived penicillin. The synthetic penicillins are produced by
enzymatic hydrolysis of 6APA by penicillin acylase enzyme and then addition of desired
side chain by chemical means,
▪ B-lactumthiozolidine ring of penicillin is constructed from l-cystine and l- valine. These
two amino acids when combined with L-α-aminoadipic acid (α-AAA) the tripeptide is
formed which undergoes two step cyclization process to give isopenicillin.

Regulation of penicillin production:

▪ The amino acids lysine is synthesized from a pathway that involves L-α- AAA, so that
penicillin and lysine share a common but branched biosynthetic pathway. Higher
concentration of lysine causes feed back inhibition of homocitrate synthase, an enzyme
involved in α-AAA synthesis. Either lysine level should keep low or α-AAA level
shouldadded during fermentation.
 ▪ Penicillin biosynthesis is affected by Po4—concentration and also shows a distinct
catabolic repression by glucose. Therefore, either slowly metabolizable sugars such as
lactose is used or fed continuously with glucose with small dose.

Penicillin Production process:

▪ Penicillin production is previously achieved by surface process ie. Solid state
fermentation and surface liquid ferementation. Now a days acommercial production is
carried out by fed batch process
▪ Inoculum (Organism): Penicilliumchrysogenum (improved strain)

i. Inoculum preparation:
▪ For inoculum preparation, spore from heavily sporulated working stocks are suspended
inwater or non-toxic wetting agensts (sodium sulfonate 1: 10000)
▪ Theses spore are then added to flask containing wheat bran and nutrient solution for
heavy sporulation
▪ Incubate for 5-7 days at 24C
▪ Spore are then transferred to seed tank and incubated for 24-48 hours at 24C with aeration
and agitation for sufficient mycelial growth
▪ These mycelia can be used for production fermenter

ii. Production fermentation:

▪ Method: fed-batch or batch
▪ Substrate: glucose, phenoxyacetic acid (fed component used for production of side
chain), Corn steep liquor, Additional nitrogen source ie,soyameal, yeast extract, Lactic
acid, inorganic ions, growth factors
▪ Fermenter: stirred tank or air lift tank
▪ pH: set at 5.5 t0 6.0 which increased upto 7-7.5 (optimum) due to liberation of NH3 gas
and consumptionof lactic acid. If pH is 8 or more, CaCO3 or MgCO3 or phosphate buffer
is added
▪ temperature: 25-27 C
▪ aeration: 0.5-1 vvm (initially more, latter less O2 )
▪ agitation: 120-150 rpm)
▪ time: 3-5 days
▪ antiform: edible oil (0.25%)

iii. Product recovery:

▪ harvest broth from fermenter tank by filtration (rotary vaccum filtration)
▪ chill to 5-10 C (because penicillin is highly reactive and destroyed byalkali and
enzyme)
 ▪ acidify filtrate to pH 2.0-2.5 with H2SO4 ( to convert
penicillin to its anionic form)
▪ extract penicillin from aqueous filtrate into butyl acetate or
amyl acetate (at this very low pH as soon as possible in
centrifugal counter current extractor)
▪ discard aqueous fraction
▪ allow the organic solvent to pass through charcoal to remove
impurities and extract penicillin from butylacetate to 2%
aqueous phosphate buffer at pH 7.5
▪ acidify the aq. Fraction to pH 2-2.5 with mineral acid and re-
extractpenicillin into fresh butylacetate ( it concentrated upto
80-100 times)
▪ add potassium acetate to the solvent extract in a
crystallization tank tocrystalize as potassium salt
▪ recover crystal in filter centrifuge
▪ sterilization
▪ further processing
▪ packaging

Application of penicillin:
▪ clinical uses of penicillin:
▪ naturally effective antibiotics against gram + bacteria
▪ used for treatment of bacterial endocarditis