Industrial production: ethanol, amylase, citric acid, single cell

protein and saffron

Alcohol: Ethanol
Microbial production of one of the organic feed stocks from plant substances such as
molasses, is presently used for ethanol production. This alcohol was produced by
fermentation in the early days, but for many years by chemical means through the catalytic
hydration of ethylene. In the modern era, attention has been paid to the production of
ethanol for chemical and fuel purposes by microbial fermentation. Ethanol is produced by
using sugar beet, potatoes, corn, cassava, and sugar cane.

Steps involved in the conversion of biomass to ethanol:

●​ Polymeric substrates broken down to monosaccharides by physical, chemical or
enzymatic techniques.
●​ Microbial fermentation converts sugar to ethanol.
●​ Alcohol obtained recovered by distillation methods to obtain as a constant boiling
mixture of 95.6% ethanol and 4.4% water by volume.
●​ Further distillation processes are required to obtain anhydrous ethanol.

STAGE I: FROM BIOMASS TO FERMENTABLE SUGARS

Sugars: Sugarcane and sugar beet contain 20% sucrose by weight. These are mechanically
crushed or after stripped and pulping the beet. Yeast produces the enzyme Invertase, which
hydrolyzes sucrose into glucose and fructose, which are later fermented by yeast cells.

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Starch: In the U.S., corn starch is the major feedstock. Dry corn is milled and added with
water to obtain a slurry. This slurry is then heated to hydrolyze soluble starch into soluble
amylose and insoluble amylopectin. Amylases and amyloglucosidases secreted by microbes
convert starch polymer into glucose.

Cellulose: Wood, agricultural waste, municipal solid waste, etc., contains the major
component lignocellulose. Lignocellulose is made of lignin, cellulose (major part), and
hemicellulose.
Steam explosion technique: Small wood chips are charged with steam in a heated
pressurized vessel at 500° F and maintained for 20 sec. Contents are rapidly decompressed,
thus solubilising hemicellulose, lignin. Cellulose is then separated by any one of the methods
given: Lignin can be extracted in high yield by treating with methanol, or dilute NaOH is
added before cellulose is degraded. Cellulose, when treated with hydrolytic enzymes, yields
glucose, while lignin, being insoluble, can be removed by filtration.
Ball milling process: Extensive physical disruption of wood followed by enzymatic
degradation of cellulose. Lignocellulosic waste is fragmented by the milling process and
suspended in water to obtain a slurry. A mixture of Trichoderma reesei cellulase is added to
the slurry to convert cellulose into glucose.

STAGE II : CONVERSION OF SUGARS TO ETHANOL

Soluble sugars are anaerobically fermented by bacteria, yeast and filamentous fungi.
Primarily yeast strains are employed for industrial production of ethanol. Saccharomyces
cerevisiae commonly used which converts glucose by glycolytic pathway to yield ethanol
(90-95% theoretical yield) and carbon dioxide.
Zymomonas mobilis , anaerobic gram negative flagellated bacterium are also used for ethanol
production. They lack spore and grow in an minimal medium requiring no organic
compounds. They are inhibited by salt conc.

Specifications of an ideal Yeast (S.cerevisiae, S.rouxii, Zymomonas mobilis

alcohol producer S.carlbergensis, Kluveromyces
fragilis)
Ability to ferment a broad Limited substrate range; cannot Limited substrate range; can
range of carbohydrates as utilize cellulose, hemicellulose, utilize glucose, fructose, and
substrate disaccharides etc., sucrose
Ethanol tolerance Toxic to yeast cells at a Tolerant to high conc. of
concentration. range 8-18% ethanol.
depending upon strain and
metabolic state. But some
strains are tolerant.

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 Low level of by-products Low level observed Liberate high-level
by-products such as
glycerol, dihydroxy acetone.
Osmotolerant Extremely osmotolerant Extremely osmotolerant
Temperature tolerant Shows metabolic rate up to an Metabolic rate at a range
optimum at 35°C but decreases 38-40°C
beyond.
High cell viability for cell Viable cells obtained for Viable cells obtained are less
recycling repeated recycling
Flocculation and Cells stick together as the wall Not exhibited.
sedimentation properties to protein binds to the mannan of
facilitate cell recycling another cell. Clumps formed
sediment rapidly, which can be
removed from the fermentation
mixture for recycling

STAGE III : ETHANOL RECOVERY

Principle of sequential distillation used which consist of cylindrical distillation column
divided into series of chambers by perforated plates. Ethanol-water mixture is boiled using
steam and vapour rises into column. Essentially each chamber of column function as
distillation unit such that proportion of alcohol goes on sequentially increasing as it rises to
the upper chamber of the column. For further purification, small amount of benzene added to
95% ethanol, which is then distilled. This distillate is nearly 100% ethanol and benzene can
be recovered.

Simultaneous Saccharification and Fermentation

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Both hydrolysis of substrate and fermentation carried out in a single vessel i.e., hydrolyzing
enzymes added to substrate and yeast cells. This process drastically reduces the production
costs. Ex., If partially hydrolyzed corn syrup is the substrate, glucoamylase enzyme (from
Aspergillus sp.,) are added to the syrup and yeast cells for ethanol production.

Clostridial fermentation:
C. thermocellum, C. thermosaccharolyticum, C. thermohydrosulfurium are thermophilic,
gram positive, anaerobic bacteria which are the candidate organisms for 1-step conversion
process. Produce extracellular cellulase enzyme which function for direct conversion of
cellulosic biomass to ethanol. Process is cheaper and eliminates need of second organism
and feedstock to support it. But major limitation is large amount of organic acids producing
hydrogen sulphide as by-product. Moreover ethanol tolerant is much lower than yeast and
Zymomonas.

Advantages of ethanol as biofuel:

●​ Renewable fuel made from plant waste.
●​ Ethanol blends can be used in all petrol engines without any modification.
●​ Completely biodegradable, hence eco-friendly.
●​ Usage significantly reduces harmful exhaust emissions such as reducing CO level to
an considerable amount.
●​ Stillage waste produced as by-product of ethanol recovery used as feedstock or as
fertilizer.
●​ Raw materials used are abundant and replenishable.

ORGANIC ACID :​
CITRIC ACID PRODUCTION

Citric acid (C₆H₈O₇, 2–hydroxy–1,2,3–propane tricarboxylic acid), a natural constituent and

common metabolite of plants and animals. Most versatile and widely used organic acid in
the field of food (60%) and pharmaceuticals (10%). Entire production is carried out by
fermentation. Citric acid is mainly used in food industry because of its pleasant acid taste
and its high solubility in water. It is worldwide accepted as “GRAS” (generally
recognized as safe), approved by the Joint FAO/WHO Expert Committee on Food Additives.

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 A. niger and certain yeasts such as Saccharomycopsis sp. are employed for commercial
production. Advantages of using this microorganism are: (a ) its ease of handling (b) its
ability to ferment a variety of cheap raw materials, and (c) high yield.

The two principal methods of selecting populations, namely, “the single-spore technique”
and the “passage method” have been used for selecting citric acid producing
microorganisms. The most employed technique to improve citric acid producing strains has
been by inducing mutations in parental strains using mutagens. Among physical mutagens,
γ-radiation have often used. To obtain hyper-producer strains, frequently UV treatment could
be combined with some chemical mutagens, e.g. aziridine, N-nitroso-N-methylurea or ethyl
methane-sulfonate.

Substrate used for fermentation

Several attempts have been made to produce citric acid using molasses, which is preferred
due its low cost and high sugar content (40-55%). Both cane and beet molasses are suitable
for citric acid production. However, beet molasses is preferred due to its lower content of
trace metals. Various other agro-industrial residues such as apple pomace, cassava bagasse,
coffee husk, wheat straw, pineapple waste, sugar beet cosset, kiwi fruit peel, etc. have been
investigated with solid state fermentation techniques for their potential to be used as
substrates for citric acid production.

Liquid fermentation​
SUBMERGED FERMENTATION​

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The submerged fermentation (SmF) process is the commonly employed technique for citric
acid production. It is estimated that about 80% of the world's production is obtained by SmF.
Several advantages, such as higher yields and productivity, and lower labour costs. Two
types of fermenters, conventional stirred fermenters and tower fermenters, are employed,
although the latter is preferred due to the advantages it offers on price, size and operation.

In SmF, different kinds of media are employed, such as sugar and starch-based media.
Molasses and other raw materials demand pre-treatment, addition of nutrients, and
sterilization. Inoculation is performed either by adding a suspension of spores or
pre-cultivated mycelia. When spores are used, a surfactant is added to disperse them in the
medium. For pre-cultivated mycelia, an inoculum size of 10% of fresh medium is generally
required. Normally, submerged fermentation is concluded in 5 to 10 days, depending on the
process conditions. It can be carried out in batch, continuous, or fed batch systems, although
the batch mode is more frequently used.

Solid-state fermentation​

Solid-state fermentation (SSF) has been termed as an alternative method to produce citric
acid from agro-industrial residues. Citric acid production by SSF (the Koji process) was first
developed in Japan and is the simplest method for its production. SSF can be carried out
using several raw materials.
Generally, the substrate is moistened to about 70% moisture, depending on the substrate's
absorption capacity. The initial pH is normally adjusted to 4.5-6.0, and the temperature of
incubation can vary from 28 to 30°C. The most commonly organism is A. niger. However,
there have also been reports with yeasts. One of the important advantages of the SSF
process is that the presence of trace elements may not affect citric acid production so
harmfully as it does in SmF. Consequently, substrate pre-treatment is not required.
Vandenberghe et al. (1999) used Erlenmeyer flasks and glass columns for the production of
citric acid from gelatinized cassava bagasse. Higher yields were obtained in flasks without
any aeration, and very little sporulation was observed.The same yields were found in column
reactors only with variable aeration. This showed great perspective to use the SSF process
for citric acid production in simple tray-type fermenters.

FACTORS AFFECTING CITRIC ACID PRODUCTION​

Medium and its components​

The presence of easily metabolized carbohydrates; sucrose was the most favourable carbon
source, followed by glucose, fructose and galactose. Directly influenced by the nitrogen
source, ammonium salts are preferred, e.g., urea, ammonia

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Aeration has been shown to have a determinant effect on citric acid fermentation (Rohr et al.,
1983; Dawson et al., 1986). Increased aeration rates led to enhanced yields and reduced
fermentation time. High aeration rates lead to high amounts of foam, especially during the
growth phase. Therefore, the addition of antifoaming agents and the construction of
mechanical “defoamers” are required.

PRODUCT RECOVERY
The recovery of citric acid from liquid fermentation is generally accomplished by three basic
procedures: precipitation, extraction, and adsorption/absorption (mainly using ion exchange
resins). Citric acid extracted by this method has been recommended for use in food and
drugs.
Precipitation is the classical method, performed by the addition of calcium oxide hydrate
(milk of lime) to form the slightly soluble tri-calcium citrate tetrahydrate. The precipitated
tri-calcium citrate is removed by filtration and washed several times with water. It is then
treated with sulphuric acid forming calcium sulphate, which is filtered off.
Mother liquor containing citric acid is treated with active carbon and passed through cation
and anion exchangers. Several anion-exchange resins are commercially available. Finally,
the liquor is concentrated in vacuum crystallizers at 20-25°C, forming citric acid
monohydrate. Crystallization at temperatures higher to this is used to prepare anhydrous
citric acid.

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 Amylase Enzyme production

Industrial Application of Microbial source Role of enzyme

α- amylase
Starch conversion B.amyloliquefaciens, Hydrolysis of starch into
B.licheniformis dextrins forming a less
viscous starch suspension
Bakery Industry B.stearothermophilus Converting starch in dough
to smaller fermentable
sugars
Detergent Industry B.licheniformis Digests starch containing
foods to water soluble
dextrin
Textile Industry Bacillus sp Used in removal of starch
sizing agent from woven
fabric

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 Fuel alcohol Production E.coli, B.subtilis Converting starch in to
smaller fermentable sugars
which are acted upon by
yeast to produce alcohol
​
α-Amylase (E.C.3.2.1.1), a hydrolase enzyme that catalyses the hydrolysis of internal
α-1, 4-glycosidic linkages in starch to yield products like glucose and maltose. It is a
calcium metallo-enzyme, i.e., it depends on the presence of a metal cofactor for its activity.
There are 2 types of hydrolases: endo-hydrolase and exo-hydrolase. Endo-hydrolases act on
the interior of the substrate molecule, whereas exo-hydrolases act on the terminal
non-reducing ends. Hence, terminal glucose residues and α-1, 6-linkages cannot be cleaved
by α-amylase. The substrate that α-amylase acts upon is starch. Applications include the
production of glucose and fructose syrup from starch

β-Amylase (EC 3.2.1.2) is an exo-hydrolase enzyme that acts from the non-reducing end of a
polysaccharide chain by hydrolysis of α-1, 4-glucan linkages to yield successive maltose
units. Since it is unable to cleave branched linkages in branched polysaccharides such as
glycogen or amylopectin, the hydrolysis is incomplete and dextrin units remain. Primary
sources of β-Amylase are the seeds of higher plants and sweet potatoes. During the ripening
of fruits, β-Amylase breaks down starch into maltose, resulting in the sweetness of ripened
fruit. The optimal pH of the enzyme ranges from 4.0 to 5.5. β-Amylase can be used for
structural studies of starch and glycogen molecules produced by various methods. In the
industry, it is used for fermentation in the brewing and distilling industries. Also, it is used to
produce high maltose syrups
γ-Amylase (EC 3.2.1.3 ) cleaves α(1,6) glycosidic linkages, in addition to cleaving the last
α(1,4) glycosidic linkages at the non-reducing end of amylose and amylopectin, unlike the
other forms of amylase, yielding glucose. γ- amylase is most efficient in acidic environments
and has an optimum pH of 3.

Production of α-Amylase
Major reasons for the increasing interest in microbial sources:
●​ Growth of microorganisms is rapid and this will in turn speed up the production of
enzymes.
●​ Easily manipulated with strain improvement using genetic engineering or mutation, or
other methods, by which the production of α-Amylase can be optimized.
●​ Tailored to cater to the needs of growing industries and to obtain enzymes with
desired characteristics like thermostability.

The most widely used source among the bacterial species: Bacillus spp., B.
amyloliquefaciens, and B. licheniformis. Other species which have been explored include

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B.cereus and B. subtilis. α-Amylases produced from Bacillus licheniformis, Bacillus
stearothermophilus, and Bacillus amyloliquefaciens show promising potential in several
industrial applications in processes such as food, fermentation, textiles and paper industries

Fungal sources of α-Amylase are confined to terrestrial isolates, mostly to Aspergillus species
and to only few species of Penicillium, P. brunneum being one of them. Penicillium
chrysogenum was used as the microbial source for producing amylase by solid state
fermentation using various substrates such as, corncob leaf, rye straw, wheat straw and wheat
bran. Among strains of Aspergillus sp. Aspergillus oryzae, A. niger and A. awamori are most
commonly used species for commercial production.

There are mainly two methods that are used for the production of α-Amylase on a
commercial scale. These are:
1) Submerged fermentation (traditional method)
2) Solid State fermentation
Submerged fermentation (SmF) employs free-flowing liquid substrates, such as molasses and
broths. Products secreted into the fermentation broth. Substrates are utilized quite rapidly;
hence the substrates need to be constantly replenished. Suitable for microorganisms such as
bacteria that require high moisture content for their growth. Used for the extraction of
secondary metabolites that need to be used in liquid form. This method has several
advantages. Utilization of genetically modified organisms to a greater extent than SSF.
Sterilization of the medium and purification process of the end products can be done easily.
Control of process parameters like temperature, pH, aeration, oxygen transfer and moisture
can be done conveniently

Solid state fermentation is a method used for microbes which require less moisture content
for their growth. The solid substrates commonly used in this method are bran, bagasse, and
paper pulp. The main advantage is that nutrient-rich waste materials can be easily recycled
and used as substrates. Simpler equipments, higher volumetric productivity, higher
concentration of products and lesser effluent generation

Purification methods commonly employed are precipitation, chromatography and

liquid-liquid extraction depending on the properties of the enzyme desired. A combination
of the above methods is used in a series of steps to achieve high purity.

The crude extracellular enzyme sample can be obtained from the fermented mass by filtration
and centrifugation. In the case of intracellular enzymes, raw corn starch may be added

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followed by filtration and subsequent steps. The crude amylase enzyme can be precipitated
and concentrated using ammonium sulphate precipitation or organic solvents. The
precipitated sample can be subjected to dialysis against water or a buffer for further
concentration. This can be followed by any of the chromatographic techniques like ion
exchange, gel filtration and affinity chromatography for further separation and purification of
the enzyme.

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 Single-cell protein

Single Cell Protein is the dried cells of microorganisms consumed as a protein

supplement by humans or animals. The protein is derived from cells of microorganisms
such as yeast, fungi, algae, and bacteria, which are grown on various carbon sources for
synthesis.
SCPs contain vitamins, e.g., thiamine, riboflavin, pyridoxine, nicotinic acid, pantothenic acid,
folic acid, biotin, cyanocobalamin, ascorbic acid, β-carotene, and α- α-tocopherol;
essential amino acids, represented by lysine and methionine; minerals; nucleic
acids and lipids.
Microorganisms should have the following properties: Absence of pathogenicity and toxicity,
protein content and quality, digestibility and organoleptic qualities, growth rate, adaptability
to unusual environmental conditions such as pH, temperature, and mineral concentrations,
and ability to utilize carbon and nitrogen sources.

Microalgae Yeast and Fungi Bacteria

phototrophic organisms. They can be divided into
unicellular yeast and mold.
They are potential SCP due Yeasts are mainly used in Bacteria are potential SCP
to their chemical aquaculture as it is the as they possess high protein
composition which contains protein-rich ingredient in content (50-80%) along with
proteins, essential fatty acids aquafeeds, with crude vitamins, phospholipids, and
mainly omega-3 fatty acids, protein contents of 38–52%. other functional molecules.
and several bioactive Mold is found to be highly Capable of growing on a
compounds. They have digestible by fish. wide range of substrates
relatively low nucleic acid from carbohydrates to
content (3–8%). gaseous and liquid
hydrocarbons
Advantages: Simple yeasts include a high level use of a wide range of
cultivation, effective of malic acid content, can substrates, their short time
utilization of solar energy, grow in acidic pH and is for generation, production of
faster growth, and high easy to harvest. vitamins and micronutrients.
protein mold include high nucleic
content of up to 10%.
disadvantage is the possible presence of the palatability issues, high
economical limitations of toxin, slower growth rate, content of nucleic acid, and
scale-up, digestibility (need and lesser content of protein production of toxins.
for cell wall disruption to (45- 65%).
release nutrients), the large
surface area needed for
cultivation, and
contamination risk in an
open pond.

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SCP Production
I. Selection of substrate and strain:
The fast-growing microorganisms are selected that are rich in protein content and possess
suitable growth characteristics. Suitable substrates are chosen that are essential for the growth
of selected microorganisms.

II. Fermentation
The inoculated medium is then placed in a fermenter where the microorganisms grow and
multiply. The conditions inside the fermenter, such as temperature, pH, and oxygen levels,
are carefully controlled to optimize the growth of the microorganisms.
III. Harvesting
After fermentation, the microbial cells are harvested and separated from the growth medium.
However, the harvesting and purification after production of SCP production remain a
problem.
IV. Post-harvest treatment
The harvested microbial cells are then dried to preserve them and reduce their volume,
making them easier to store and transport. SCP processing for food The dried cells are then
processed to remove impurities, improve their nutritional content, and enhance their flavor
and texture.

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Advantages:
●​ Microorganisms grow at a faster rate compared to the growth of protein-rich grain
which takes a year for production.
●​ The quality and quantity of protein are better (60-80%).
●​ A wide range of inexpensive raw materials can be used easily.
●​ The production process is easy and simple.
●​ The microorganisms can be easily subjected to genetic manipulation.
●​ The microorganism can be produced all-around a year.
●​ They can utilize a wide range of substrates.
●​ The production of SCP is eco-friendly, cost-effective, and energy efficient.

Disadvantages
●​ The production of SCP is a complex process that requires strict control over various
environmental factors.
●​ Maintaining the quality of SCP is a hard job as the harvesting and purification after
production of SCP production remains a problem.
●​ SCP is not a suitable source of all the essential amino acids, so it is typically used as a
supplement to other protein sources
●​ Despite the many potential benefits of SCP, consumer perception remains a significant
challenge, as many people may be skeptical of consuming a product derived from
microorganisms.

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COMMERCIAL PRODUCTS

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