Institute of Life Long Learning

Applications of microbes in industry: Production of
primary and secondary metabolites
Lesson: Applications of microbes in industry:

Production of primary and secondary metabolites
Author Name: Dr. Simran Jit & Dr. Nidhi Garg
Reviewer: Dr. R.K.Gupta
College/ Department: Miranda House , Deshbandhu
College, University of Delhi
Institute of Life Long Learning Page 0

Table of Contents
 Introduction
 Primary metabolites
 Secondary metabolites
 Overview of fermentation process
 Production of:
 Antibiotics
 Penicillin
 Streptomycin
 Amino acids
 Lysine
 Glutamic acid
 Organic acid
 Acetic acid
 Lactic acid
 Summary
 Exercise/ Practice
 Glossary
 References/ Bibliography/ Further Reading

Introduction
Industrial fermentation based on the end-product application can be categorized into four types:
1. Biomass: The end-product is viable cellular material eg, single cell protein, baker’s yeast,
probiotic cultures.
2. Extracellular metabolites: Chemical compound intermediates of microbial biochemical
pathways are produced and can be divided two groups:
a. Primary metabolites (produced during the growth phase of the organism, eg, ethanol, citric
acid, glutamic acid, lysine, vitamins and polysaccharides)
b. Secondary metabolites (produced during the stationary phase, eg, penicillin, cyclosporin A,
gibberellin, and lovastatin).
3. Enzymes and other proteins (intracellular components): A key component of this process is
lysis of cells at the end of fermentation. Proteins are typical end products and need to be
purified and crystallized.
4. Substrate transformations: Raw material is biologically transformed into a finished product.
Generally used for steroid transformations, food fermentations and sewage treatment.
This chapter focuses on applications of microbes for industrial production of primary and
secondary metabolites. Metabolism in microorganisms involves two pathways: Primary
metabolic pathways (PMPs) produce too few end products while secondary metabolic pathways
(SMPs) produce a variety of products (Fig 1).
There are some similarities between the pathways that produce primary and secondary
metabolites, namely that the product of one reaction is the substrate for the next and the first
reaction in each case is the rate-limiting step. Also the regulation of secondary metabolic
pathways is interrelated in complex ways to primary metabolic regulation.
Figure 1: Production of primary and secondary metbolites.

Source: Author, ILLL in house

Primary metabolites
 Involved in growth, development and reproduction. Hence, essential

for survival and existence of the organism and reproduction.
 Formed at the same time as new cells.
 Production curve follows the growth curve.
 Formed in trophophase during exponential growth as normal end
products of primary metabolism.
 Also called central metabolites as these maintain normal
physiological processes.
 Cells maintain optimum concentration of all macromolecules
(proteins, DNA, RNA etc.).
 Produced in adequate amount to sustain cell growth for example
vitamins, amino acids, nucleosides etc.
 Overproduction can be genetically manipulated. Auxotrophic (auxo,
“increase,” and trophos, ‘‘food’’) mutants having a block in steps of a
biosynthetic pathway for the formation of primary metabolite (Fig 2).
 Growth rate slows down due to limited supply of any other nutrient.
Metabolism does not stop but product formation stops.
 Industrially important for example ethanol, acetone, lactic acid, CO2.
 Common food supplements, L-glutamate and L-lysine, are produced
and purified via the mass production Corynebacterium glutamicum.
 Citric acid, commonly used in pharmaceutical and cosmetic industries
is produced by Aspergillus niger.
Figure 2: Over production of primary metabolite. To maximize

production manipulation of feedback inhibition pathways is performed. Another
approach is to use auxotrophic mutant with defective metabolite production.

Secondary metabolites
• Secondary metabolites are not produced until the microbe has largely completed its
logarithmic growth phase and entered the stationary phase of the growth cycle.
Period of production is called idiophase and metabolites as idiolites.
• In the idiophase, cells do not divide but are metabolically active.
• Idiolites are organic compounds produced only after considerable number of cells
and a primary metabolite have accumulated (end or near the stationary phase of
growth). Rather it can be said that these are produced under sub-optimal
concentrations of O2, deviations of pH or when primary nutrient source is depleted.
• Though idiolites are a characteristic feature of fungal, yeast, actinomycetes and
bacterial growth but are not produced by a few strains of E. coli.
• In some strains secondary metabolite are produced by further conversion of a
primary metabolite.
• Alternatively, these may be metabolic products of the original growth medium.
• Not necessary for growth, development, and reproduction like primary metabolites.
Their production is influenced by environmental factors.
• Secondary metabolites are synthesized for a finite period by cells that are no longer
undergoing balanced growth. A single microbial type can produce very different
metabolites.
• Their production is regulated by complex biochemical pathways and some strains
can produce a variety of idiolites. For example a strain of Streptomyces can produce
a variety of 35 anthracyclines.
• Typical examples include antibiotics, toxins and pigments to name a few.
• Bacitracin, a peptide antibiotic, derived from Bacillus subtilis, is commonly used as
a topical drug. Bacitracin is synthesized in nature via non-ribosomal peptide
synthetase (NRPSs). It has a wide spectrum against Gram positive and Gram
negative bacteria. Non-essential for growth.
• Overproduction of secondary metabolites can be achieved by manipulating larger
number of genes (gene cassettes).

Figure 3: Primary and secondary metabolism. During the trophophase, the cell mass
increases logarithmically but as the resources become limiting growth rate drops and production stops.
Idiolites are special metabolites usually possessing bizarre chemical structures and although not
essential for the producing organism's growth in pure culture, they have survival functions in nature.
Figure 4: Scheme for industrial production of metabolites.

Industrial Production of Microbial Metabolites

The process of industrial production of microbial metabolites can summarized as in Fig 4. Basic
steps are:
1. Screening, selection, maintenance of source microorganism for the production of target

metabolite (primary or secondary).
2. Optimization and standardization of growth medium and conditions (w.r.t. choice of
fermenter vessel, aeration, temperature, agitation, pH, etc.) for large-scale (fermentation)
protocol.

Preparation of the microorganism and the raw materials required for the
microorganism to grow and produce the desired product is called upstream
processing.
3. Sterilization of the medium, fermenter and ancillary equipment.

4. Active, pure culture in sufficient quantities is used to inoculate growth medium in fermenter.
5. The growth of the organism in the production fermenter under optimum conditions for
product formation.
Growth of the target microorganism in a large bioreactor (usually >100 litres) with
the consequent production (biotransformation) of a desired compound is the phase of
fermentation and transformation.
6. The extraction of the product and its purification.

7. Disposal of effluents produced by the process.
Purification of the desired compound from either the cell medium or the cell mass is called
downstream processing.
Production of antibiotics
Penicillin
 This antibiotic was first discovered by Alexander Fleming in 1929 and the producing organism
associated with its production was Penicillium notatum. Penicillin (PCN or pen) is the one of
most widely used effective antibiotics and is now commercially produced from Penicillium
chrysogenum mutant strains NRRL-1951, Wisconsin 54-1255 and AS-P-78.
 The penicillin produced without addition of any side-chain precursors are referred to as
natural penicillin (Penicillin G (intravenous use) and V (oral use)) (Fig 5).
 The conventional penicillin has been replaced by newer versions eg. Ampicillin, methicillin,
carbenicillin (See Fig 9).
 The basic structure of the penicillin is 6-aminopenicillinic acid (6-APA) which consists of a
thiazolidine ring with a condensed β-lactam ring. Depending upon the acyl moiety at position
6 there are various penicillins.
 The acyl group to be attached to the ring is either added in the growth medium producing
biosynthetic penicillins or appended chemically to 6-APA obtained by deacetylation of
penicillin through acylases forming semi-synthetic penicillins.

Figure 5: Structure and Function relationship of Penicillin:

Source: Yusof M, Mei Yi DC, Enhui JL, Mohd Noor N, Wan Ab Razak W, Saad Abdul Rahim A. An
Illustrated Review on Penicillin and Cephalosporin: An Instant Study Guide for Pharmacy
Students. WebmedCentral PHARMACEUTICAL SCIENCES 2011;2(12):WMC002776 doi:
10.9754/journal.wmc.2011.002776.

Value addition: Did you Know?
Heading text: Action of penicillin

Diagram depicting formation of cross-links in the bacterial cell wall by a penicillin

binding protein (PBP, an enzyme) and subsequent suicide inhibition by penicillin. 1.
The bacterial cell wall consists of strands of repeating N-acetylglucosamine (NAG) and N-
acetylmuramic acid (NAM) subunits. The NAM subunits have short peptide chains attached to
them. (The exact composition of these can vary. The proximal alanine is usually L-ala and
the distal two are usually D-ala.) 2. The PBP binds the peptide side chains and forms the
cross-link with the expulsion of one D-Alanine from one peptide side chain. 3. The PBP
dissociates from the wall once the cross-link has been formed. 4. When penicillin is
administered to the cell in the culture medium, it enters the active site of the PBP and reacts
with the serine group which is important in its enzymatic activity. 5. The beta-lactam ring of
penicillin (represented here as the top of the "P") is irreversibly opened during the reaction
with the PBP. Penicillin remains covalently linked to the PBP and permanently blocks the
active site. As a result PBP is unable to bind the NAG and NAM sites due to penicillin
occupied sites hence no cross linking takes place and cell wall remains porous.
Source: https://commons.wikimedia.org/wiki/File:Penicillin_inhibition.svg

Value addition: Did you Know?

Heading text: Discovery of ‘Mould Broth Filtrate’-First antibiotic
Body text: Alexander Fleming discovered mould growing on a bacterial culture in the
1920s and Dr. E.B. Chain and Sir Howard Florey worked as a team that brought
curative power to millions. For this discovery all three were conferred Nobel Prize in
1942 in Medicine and Physiology.
Source: https://www.youtube.com/watch?v=VGC5JOLQoGo
 Penicillin inhibits the cell wall synthesis in growing bacterial cells by binding with penicillin-
binding protein (PBP) of the bacterial cell wall involved in the trans-peptidation reaction
(cross linking) of the peptide side chains of the adjacent peptidoglycan in the bacterial cell
wall.
 Due to the defective cell wall, the bacteria are unable to withstand the osmotic shocks and
are ultimately lysed.
Production of Penicillin
 In general, commercial penicillin is produced by fed-batch fermentation process using

bioreactors of 30K-250K capacity. The fermentation temperature is maintained around 25-
27C, pH 6.5-7.0 with a constant supply of oxygen.
 The fermentation medium is composed of a carbon source like lactose and a nitrogen source
like corn steep liquor. The media used for the production of penicillin contains corn steep
liquor solids 3.5%, lactose 3.5%, glucose 1%, calcium carbonate 1%, potassium di-hydrogen
phosphate 0.4%, edible oil 0.25% and penicillin precursors (Fig 6). Additional supplements
like yeast extract, soy meal, ammonium salts are also added. In addition, side chain
precursors like phenyl acetic acid or phenoxyacetic acid are also added to the medium.

 Corn steep liquor is an important ingredient in the broth; it supplies the fungus with nitrogen
and growth factors.
 Since high levels of glucose repress penicillin production, lactose (from whey) is added to the
corn steep liquor in large amounts as a carbon source.
Figure 6: Summary of penicillin production and recovery.

 The media is placed at 25-27ºC with pH maintained at around 5.5-6.0.

 Lyophilized spores of Penicillium are used as inoculum. For better production of penicillin,
development, of loose mycelial pellets is however preferred.
 The penicillin fermentation process can be divided into two phases: a vegetative growth
phase (around 40 h) followed by penicillin production phase (150-180 h). (Fig 7)

1. Growth phase. The growth phase lasts for about 40h during which the cell mass increases
very rapidly and oxygen demand is very high.
2. Penicillin production phase. The biomass production is greatly reduced and rate of
penicillin production increases. As the lactose approaches limiting levels and cell densities
in the fermenter become very high, addition of low levels of glucose maximize penicillin
yield. Various other media components are fed during this phase to extend the penicillin
production for 120-180h.
Figure 7: Production of Penicillin. During the growth phase, very little penicillin is
produced, but once the carbon source has been nearly exhausted, the penicillin production
phase begins. By supplying additional carbon and nitrogen at just the right times, the
production phase can be extended for several days.
Recovery of penicillin
 This process is referred to as downstream processing. For this, the fermentation broth is first
chilled and passed through rotary filter to remove biomass (Fig 6).
 The filtrate is acidified to pH 2.0-2.5 with H2SO4 and the penicillin is extracted with butyl
acetate.
 Penicillin is extracted from solvent (butyl acetate) into aqueous buffer with pH 7.0.
 Aqueous fraction is acidized again and re-extracted into butyl acetate.
 To recover penicillin, the extract is crystallised by addition of sodium or potassium hydroxide
or acetate. The penicillin GK+ salt crystals are washed with acetone by centrifugation and
dried under vacuum. The penicillin crystals obtained are more than 90% pure.
Semisynthetic penicillins
The unstable nature of β-lactam ring makes penicillin molecule susceptible to metabolism. Thus,
one of the major drawbacks is that penicillin is not orally active and must be injected. The
present drug development focuses on i) to increase chemical stability for oral administration; ii)

to increase resistance to β -lactamases and iii) to increase the range of activity. Analogs of
penicillin are produced by varying the carboxylic acid during fermentation or by the semi-
synthetic approach (Fig 9). These analogs have increased stability and are effective against wide
range of pathogens.
Semi-synthetic penicillins are broad-spectrum antibiotics and most of them can be taken orally
and thus do not require injection. Natural penicillin is treated to yield 6-APA which is further
modified chemically by the addition of a side chain (Fig 8).
Figure 8: Derivation of natural penicillin to produce semisynthetic and

biosynthetic variants.

Figure 9: Analogs of Penicillin.

Streptomycin
 Streptomycin is a broad spectrum antibiotic (antimycobacterial) belonging to oligosaccharide

antibiotic/aminoglycoside family.
 Streptomycin was discovered by Schatz, Bugie, and Waksman in 1944 from Streptomyces
griseus isolated from soil.
 It was the first effective treatment against Mycobacterium tuberculosis. It is also effective
against some Gram positive bacteria primarily Staphylococcus aureus and is mainly used
against the pathogenic bacteria resistant to penicillins.
 This antibiotic is also active against other diseases, e.g., plague (Pasteurella pestis),
brucellosis (Brucella abortus), tularemia Francicella tularieusis).
 Streptomycin is a protein synthesis inhibitor. It binds to the small 16S rRNA of the 30S
subunit of the bacterial ribosome, interfering with the binding of formyl-methionyl-tRNA to
the 30S subunit. This leads to codon misreading followed by complete or partial inhibition of
protein synthesis and eventually death of microbial cells. Humans have ribosomes which are
structurally different from those in bacteria, so the drug does not have this effect in human
cells.

Figure 10: Isolation and screening of antibiotic producers.

 The isolation and screening of novel antibiotic producer is outlined in Fig 10. The first phase
is the isolation for antibiotic producing strains for example of Streptomyces. This is done on
selective media hence most colonies obtained are of Streptomyces. When the indicator strain
is overlaid, some strains which show sensitivity as measured by Zone of Inhibition are picked
up. These colonies represent antibiotic producing strains. Common indicator strains include
Escherichia coli, Bacillus subtilis, Staphylococcus aureus and Klebsiella pneumoniae.
 Then, the producer strain colony is picked and streaked to test for its antibiotic spectrum. It
is streaked over one-third of plate and incubated. After good growth is obtained, the bacterial
species (5 in this case) are streaked perpendicular to the producing organism, and the plate
was further incubated. The failure of several species to grow near the producing organism
indicates that it produced an antibiotic active against these bacteria.
Figure 11: Structure of Streptomycin.

Source: Abdul Rahim AS, Muhamad Sayuti M, Hau KC, Ee DC, Wan Zaki W, Raskitar N, et al. An
Illustrated Review About Aminoglycosides. WebmedCentral PHARMACEUTICAL SCIENCES
2011;2(12):WMC002744 doi: 10.9754/journal.wmc.2011.002744
Production of Streptomycin

 Streptomycin is derived from the bacterium Streptomyces griseus.Streptomycin has three

constituents namely; N-methyl L-glucosamine, Streptose and streptidine (Fig 11).
 The present day streptomycin producers are the mutants of Streptomyces derived for higher
yields with yield as high as 25,000 units per ml.
 The industrial production of streptomycin is carried out using submerged fermentation
processes.
 As the Streptomyces mutants are genetically unstable, the spores are maintained as soil
stocks or lyophilized and are used for inoculating sporulation medium, which is then
transferred to germinator where biomass is increased for inoculating fermenters.
 The fermentation media consists of glucose, starch, dextrin, soy meal, corn steep liquor,
sodium sulphate. The streptomycin fermentation requires high aeration and agitation.
 The fermentation is carried out at 28-30ºC with pH maintained at 7.6-8 for good productivity.
The fermentation lasts for 5-7 days with of yield of 1-3 g/L of the fermentation broth (Fig
12).
Figure 12: Streptomycin production by S. griseus. Glucose and other

carbohydrates have been reported to interfere with antibiotic synthesis and this effect depends
on the rapid
 The streptomycin fermentation proceeds through three phases (Fig 12 and 13):
1. In first phase, the organism produces proteases which digest the soybean meal and
release ammonia and carbohydrates. These are utilized for increasing the biomass.
Glucose is slowly utilized and net production of streptomycin is low during this phase. The
pH of the medium increases from 6.7 or 6.8 to 7.5 or higher. This phase lasts for 24h.
2. The next phase is the idiophase or the stationary phase during which maximum
streptomycin (secondary metabolite) is produced. It ranges from 24h to 6-7 days. Rapid
utilization of ammonia and glucose occurs with no mycelial growth and pH during this
phase remains fairly constant at 7.6 to 9.0.
3. In the last phase (death phase) the sugars have been completely depleted in the medium
and streptomycin production ceases completely. The ammonia released due to the cell

lysis raises medium pH. Fermentation broth is generally harvested before the last phase
begins.
Figure 13: Phases of streptomycin production.

Recovery of Streptomycin
 On completion of fermentation, the mycelium is separated from the broth by filtration.

 The remaining liquid is then percolated through cation exchange resin columns where
streptomycin gets adsorbed and is finally eluted by washing with buffer as streptomycin
sulphate.
 Further impurities are removed by treating it with sodium hypochlorite, EDTA and activated
carbon.
 The purified streptomycin sulphate solution is concentrated under vacuum and dried
aseptically.

Production of amino acids
Lysine
 L-Lysine (Fig 14) is an essential amino acid as it is not synthesized by humans and hence has
to be obtained from the diet.
 Cereals and vegetables also have low lysine content and are often fortified with lysine
supplements. It is also produced for use as animal feed.
Figure 14: Structure of Lysine. Abbreviated as Lys or K, is an α-amino acid. Lysine's

codons are AAA and AAG.
Source:https://commons.wikimedia.org/wiki/File:Lysine-zwitterion-2D.png
Production of Lysine
 Lysine is produced in a two-step process by combination of two bacterial strains:

A. Lysine-histidine double auxotrophic mutant Escherichia coli and
B. Aerobacter aerogenes.
 Firstly, E. coli is grown in molasses based medium containing glycerol, corn-steep liquor, and
(NH4)2HPO4 to produce diaminopimelic acid (DAP). Apart from glycerol, ethanol or alkanes
supplemented with soybean hydrolysates can also be used as carbon sources. The
temperature is maintained at 28ºC.
 After three days of fermentation under optimized conditions of temperature and pH in
aerated stirred tank rectors, the entire fermentation broth is then incubated with Aerobacter
aerogenes at 35ºC, which decarboxylates the DAP to L-lysine.

Figure 15: Biotransformation of Diaminopimelic acid (DAP) to lysine by

DAP decarboxylase.
 Another industrial method utilizes one step single strain approach.

 For this, Corynebacterium glutamicum (homoserine auxotrpohs) and Brevibacterium
(methionine-threonine double auxotrophs) are used and production of L-lysine is controlled at
the level of the enzyme aspartokinase.
 In this method Aspartate is used as the precursor and through series of biochemical
pathways as shown in Fig 16 excess lysine feedback inhibits the activity of this enzyme.
Figure 16: Single step production of lysine using mutant

Corynebacterium glutamicum. In the biochemical pathway leading from aspartate to
lysine; lysine can feedback-inhibit activity of the enzyme aspartokinase causing cessation of
lysine production. S-aminoethylcysteine (AEC) an analog of lysine is identical to lysine in
structure except that a sulfur atom replaces a methyl (-CH2) group. AEC normally inhibits
growth, but AEC-resistant mutants of C. glutamicum have an altered allosteric site on their
aspartokinase and grow and overproduce lysine because feedback inhibition no longer occurs.
 To achieve higher production values mutants of C. glutamicum are used in which

aspartokinase is no longer subject to feedback inhibition.
 Such mutants can be generated by repeated culturing in a medium containing lysine analog
S-aminoethylcysteine (AEC). AEC binds to the allosteric site of aspartokinase and inhibits
activity of the enzyme. However, AEC-resistant mutants can be obtained easily and
synthesize a modified form of aspartokinase whose allosteric site no longer recognizes AEC or
lysine.

 In such mutants, feedback inhibition of this enzyme by lysine is nearly eliminated.

 A typical AEC-resistant mutant of C. glutamicum can produce over 60 g of lysine per litre in
industrial fermenters.
 Industrial production is carried out as batch process in aerated stirred tank reactors. A
variety of carbon sources such as sugarcane molasses, acetate, ethanol or alkanes
supplemented with soybean hydrolysates can be used.
 Gaseous ammonia or ammonium salts are used as nitrogen source and urea along with other
growth factors such as L-homoserine or L-threonine and L-methionine.
 The addition of ammonia and urea also helps in maintaining a neutral pH around 7.
 It is critical to maintain biotin content in the medium >30 μg/L for optimal lysine production
and lower concentrations result in the accumulation of L-glutamate instead of L-lysine.
 To shorten the lag phase during fermentation larger ~ 10% inoculum is used.
 Lysine production starts in the early log phase of growth and through the stationary phase
and a maximum of production 40-45 g/L lysine is achieved in 60h.
Recovery of Lysine
 Lysine is recovered by acidification of the cell free fermentation broth to pH 2.0 with
hydrochloric acid.
 This acidified mixture is then passed through cation exchange column where L-lysine gets
adsorbed in the ammonium form.
 Bound L-lysine is then eluted from column with dilute ammonia solution.
 The eluate is re-acidified and crystallized as L-lysine hydrochloride.
Glutamic acid
 Kyowa Hakko, Japan was the first company to start the production of L-glutamic acid
(monosodium glutamate, MSG) by fermentation using Corynebacterium glutamicum.
 Breviacterium, Microbacterium and Arthrobacter are some other glutamic acid producing
bacteria.
 These bacteria are Gram positive, nonsporulating, non-motile, require biotin. Importantly,
these lack or have little α-ketoglutarate dehydrogenase activity and show high glutamate
dehydrogenase activity (Fig 17).
 Isocitrate dehyderogenase catalyses the decarboxylation and dehydrogenation of isocitrate to
α-ketoglutarate in tricarboxylic acid (TCA) cycle. This α-ketoglutarate is the key precursor,
which is converted into L-glutamic acid via reductive amination with free NH4+ ions, catalysed
by glutamate dehydrogenase. The strains used for commercial production of glutamic acid
lack or have very low α-ketoglutarate dehydrogenase activity.

Figure 17: Enzymes involved in the biosysnthesis of L-glutamate

Production of Glutamic acid
 The glutamic acid bacteria in normal growth conditions synthesize glutamic acid intracellularly
and do not secrete it out of the cell because of the rigid cell wall.
 However, the permeability of the bacterial cells can be enhanced by:
i. restricting the formation of normal phospholipid biosynthesis using biotin deficient media,
ii. limiting oleic acid in oleic acid auxotrophs,
iii. limiting glycerol in glycerol auxotrophs,
iv. addition of surfactants (e.g. Tween 60) or by adding C16 - C18 saturated fatty acids,
v. addition of penicillin in case of biotin rich media to weaken the cell wall thus rendering high
permeability for secreting glutamic acid in the culture broth and
vi. limiting biotin, an essential co-factor. Deficiency of biotin affects fatty acid biosynthesis,
membrane formation falters, permeability is affected and intracellular export of glutamic acid
is altered.
 Stirred tank reactor up to 450 m3 is used for industrial production of glutamic acid. The
fermentation is carried out aerobically at 30-37°C, depending on the microorganisms used.
 Glucose, fructose, sugar cane and sugar beet molasses, and starch hydrolysates are some
carbon sources used in production of L-glutamic acid.

 Penicillin or fatty acid derivatives (e.g. Tween 60) are added in the sugar cane or sugar beet
molasses based medium upsetting the cell wall synthesis of these bacteria as these carbon
sources contain high biotin (0.02-0.12 mg/Kg) content favoring the formation of cell
membrane with high lipid content.
 Acetate, methanol, ethanol, acetaldehyde, or n-alkane have also been employed as carbon
source in the production of L-glutamic acid by bacteria, but still cane sugar molasses or
starch hydrolysates are the main carbon sources.
 Ammonium salts or ammonia are generally used as nitrogen source. In case of glutamic acid
bacteria having high urease activity, urea can also be used as nitrogen source in the medium.
 Oxygen supply is necessary for glutamic acid production and under oxygen deficiency,
excretion of lactate and succinate occurs, whereas excess oxygen results in ammonium ion
deficiency, ceasing the growth and production of α-ketoglutarate, thus lowering the L-
glutamic acid yield in both cases (Fig 18).
 Medium pH during fermentation is maintained at 7-8 by the addition of alkali/ammonia.
 L-glutamic acid starts accumulating from the mid-way of the fermentation process which
normally lasts for 30-35 h and finally L-glutamic acid level reaches to 100g/L in the
fermentation broth in case of Brevibacteriumdivaricatum. In acidic pH with excess ammonia,
glutamine is produced instead of L-glutamic acid.
 Glutamic acid bacteria convert 50-60% of the added carbon source to L-glutamic acid under
the optimal culture conditions.
Recovery of Glutamic acid

 At the end of the fermentation the broth contains l-glutamate in the form of its ammonium
salt.
 L-Glutamic acid is recovered from the fermentation broth by separating the cells from the
culture medium and passing the broth through anion exchange resin.
 L-Glutamate ions bind to the resin and ammonia is released.
 Elution is performed by NaOH to form monosodium glutamate (MSG).
 MSG crystals are then prepared and further purified to food-grade quality (Fig 19).

Figure 18: Biosynthetic production of Glutamic acid production by

Corynebacterium glutamicum. Bold arrows represent major carbon flows while the
dashed lines indicate reactions that are being used to a lesser extent. On left growth occurs by
the glyoxylate bypass to provide critical intermediates in the TCA cycle. On the right pathway
after growth is depicted. As can be seen most of the substrate carbon is processed to
glutamate.

Figure 19: An overview of L-glutamate (Mono Sodium Glutamate)

production.
Source:http://glutamicacid.wikispaces.com/?responseToken=0a62912f0da7aaf080bf8a89fb7aef
5d9
Production of Organic acid
Acetic acid (Vinegar)
 Acetic acid (CH3COOH) is the principal constituent of vinegar. It is also known as ethanoic
acid, ethylic acid, vinegar acid, and methane carboxylic acid (Fig 20).
 Glacial acetic acid is the pure compound (99.8%), as distinguished from the usual water
solutions known as acetic acid.
 Vinegar is used as a flavouring agent in salads and other foods, and because of its acidity, it
is also used in pickling. Foods pickled with high concentrations of vinegar can be stored
unrefrigerated for years.

Figure 20: Structure of acetic acid.

Source: https://commons.wikimedia.org/wiki/File:Acetic-acid-2D-flat.png
Production of Acetic acid

 Acetic acid producing bacteria are Gram-negative bacteria which belong to the family
Acetobacteriaceae, and to the alpha-subclass of Proteobacteria. The recognized genera are:
Acetobacter, Acidomonas, Gluconobacter, Gluconacetobacter, and Kozakia.
 These are obligate aerobes, motile rods that carry out the incomplete oxidation of alcohols
and sugars, leading to the accumulation of organic acids as end products. The acetic acid
bacteria are tolerant of acidic conditions and most strains thrive well at pH values lower than
5.
 Two important biochemical changes take place in the production of vinegar (Fig 21):
A. Alcoholic fermentation of a carbohydrate.
B. Oxidation of the alcohol to acetic acid.
Figure 21: Acetic acid (vinegar) production. In the first step ethanol is oxidised
to acetaldehyde by alcohol dehydrogenase (ADH). Second oxidation by the same enzyme
produces acetic acid. ADH donates electrons directly to ubiquinone (UQ) in the cytoplasmic
membranes and thus the ethanol oxidase respiratory chain of acetic acid bacteria.
 Different starting substrates eg., dilute ethanol solutions, fermented rice, fruit juices,
hydrolysed starchy material, sugar syrups, alcoholic apple juice (hard cider) are used
depending on the type of vinegar to be produced.
 The production of ethanol from a carbohydrate source is carried out at 30-32C using
anaerobic yeast Saccharomyces cerevisiae. The alcohol produced by this fermentation act as
a substrate for the second step carried out by strictly aerobic bacteria.

 The flagellated Acetobacter and Gluconobacter are principal acetic acid producing
microorganism. While Acetobacter is able to further oxidize acetic acid forms to CO2, for
Gluconobacter acetic acid is a “deadend”product (Fig 22).
A B
Figure 22: Acetic acid producing bacteria: (A) Acetobater is a Gram negative
bacterium capable of converting alcohol into acetic acid. The aerobic rod shaped bacterium
grows in temperatures around 30C and found in any alcoholic niches such as flowers, soil and
water. (B) Gluconobacter partially oxidize carbohydrates and alcohol through oxidative
fermentation. Used to synthesis vitamin C, D-gluconic acid and ketogluconic acids.
Source:http://microbiologyglossary.wikispaces.com/Acetobacter_acetihttp://www.micronaut.ch/
shop/gluconobacter/
 In the reaction depicted in Figure alcohol acts as the electron donor and is oxidised to acetic
acid by Gluconobacter.
 Thermophilic bacteria can utilize cellulose instead of alcohol as substrate while
Acetobacterium woodii and Clostridium aceticum can synthesize acetic acid from hydrogen
and carbon dioxide.
 Since aerobic microbes employed for the acetic acid production, oxygen demand is very high
during fermentation.
 Three processes are used for commercial vinegar production.
1. Open-vat method: In this process, wine is placed in shallow vats to facilitate exposure to the
air. At the surface of the liquid, the acetic acid bacteria develop as a slimy layer. The process
is continued over a period of several months at room temperature, the fruit juices ferment
into alcohol and then oxidized into acetic acid. Although this original process is still used but
it is not very efficient as the bacteria contact both the air and substrate only at the surface
(Fig 23).
Alcohol is poured into wooden barrels in which holes have been drilled on the top as well as
ends. To initiate the process a starter vinegar stock is added and the barrel is filled to a level
just below the holes on the ends. The barrel is then covered with nets and allowed to sit for
several months. The room temperature is kept at approximately 29°C.

Figure 23: Open-vat method of vinegar production. This method was used
until nineteenth century. In this method fermented fruit juices are exposed to the airborne
acetic acid producing bacteria. Thereafter the contents are removed by filtration. Since wild type
strains are used, the final extract also contains a mixture of other acids such as lactic and
propionic acid besides vinegar.
2. Trickle method or generator method: This method is used for the production of distilled and
industrial vinegar and operated in a continuous fashion. Loosely packed twigs or wood
shavings are arranged as tall oak vats or column and filled with charcoal or grape pulp. These
provide a surface for bacteria to grow to form a biofilm using trickling fluid as a substrate.
These wood shaving are not consumed and can last from 5 to 30 years, depending on the
kind of alcoholic liquid used in the process. Alcoholic liquid is trickled over the top of the vat
and slowly drips down the fillings. The process is aerated with dual oxygen supply through
punched holes at the top and perforations at bottom. A stream of air is supplied from bottom
which then passes upward to provide oxygen to aerobic bacteria. The process is continued for
several weeks after which vinegar is poured off from the bottom of vat into storage tanks.
This may be percolated again to complete oxidation of alcohol and hence maximise
production. The vat is maintained within the temperature range of 15-34C (Fig 24).

The major advantage of this method is that it is low on cost. In addition, ease of
maintenance, small space requirement and high acetic acid concentrations of the product are
other benefits of this method. However, disadvantages of the process are: long start-up time,
loss of ethanol by volatilization, and production of slime-like material by Acetobacter.
Figure 24: Production of vinegar by continuous fermentation. Alcoholic

juice is trickled through wood shavings and air is passed from the bottom and moves upward.
Acetic acid bacteria colonize the wood shavings form a biofilm that oxidizes alcohol to acetic
acid. The dilute acetic acid is pooled in the collecting chamber. It is recycled through the
generator. After the acetic acid content reaches 4% (the minimum concentration to be
labelled as vinegar), the pooled product is drained off. This submerged, batch process using
Frings acetator can produce 12% acetic acid in about 35 h.
3. Bubble method: It incorporates industrial incubation techniques to introduce and mix air into a
fermenter containing the alcoholic substrate and inoculated with acetic acid bacteria. The
bubble method is highly efficient; 90–98% of the alcohol is converted to acetic acid.
Recovery of Acetic acid
 The vinegar produced by trickle method has high acetic acid content (14%), and must be
diluted with water to bring its acetic acid content to a range of 5-6%.
 Distilled vinegar is produced by pouring the diluted liquid into a boiler and brought to its
boiling point. A vapour rises from the liquid and is collected in a condenser. It then cools and
becomes liquid again. This liquid is then bottled as distilled vinegar.

Figure 25: Some applications of acetic acid in cosmetic, food and

chemical industry.
Lactic acid
 Lactic acid (2-hydroxypropanoic acid) was discovered and isolated in 1780 by the Swedish
chemist Scheele from sour milk and the first organic acid produced microbiologically in 1881
by Charles E. Avery at Littleton, Massachusetts, USA (Fig 26 a).
 Lactic acid is a highly hygroscopic, syrupy liquid and is technically available in various grades,
i.e. technical grade, food grade, pharmacopoeia grade and plastic grade (Fig 26 b).

Figure 26 a: During intense exercise our body cannot provide enough

oxygen to allow the complete combustion of glucose to carbon dioxide.
Under these conditions, an alternative means of obtaining energy from glucose by converting it
to lactic acid.
Source:http://2012books.lardbucket.org/books/principles-of-general-chemistry-v1.0/s09-07-
end-of-chapter-material.html
Figure 26 b: Commercial applications of lactic acid.


Figure 27: Lactic acid fermentation.

Source:https://eochemistry.wikispaces.com/acid+and+running+-+alek,+adam
 Lactic acid producing bacteria (LABs) belong to the genera Lactococcus, Streptococcus,
Pediococcus, Oenococccus, Leuconostoc and Lactobacillus and have been isolated from
natural habitats like plants or fermented foods like dairy and meat products (Fig 27, 28).
 Materials frequently used include glucose syrups (e.g. derived from starch hydrolysis),
maltose-containing materials, sucrose (e.g. from molasses) or lactose (whey).
 Lactobacillus delbrueckii subsp bulgaricus which utilize lactose as substrate is industrially
important organisms used for the production of dairy products like yoghurt, cheese,
buttermilk and kefir and is also used to produce lactic acid from whey.
 Lactobacillus delbrueckii and L. pentosus produce lactic acid when glucose and sulphite waste
liquor are used as substarte, respectively.
 Other biological agents capable of producing lactic acid are also used such as strains
of Rhizopus, Escherichia, Bacillus, Kluyveromyces and Saccharomyces.

A B
Figure 28: Lactobacillus bulgaricus. (A)It is a gram-positive rod that may appear
long and filamentous. It is non-motile and does not form spores. It is regarded as aciduric or
acidophilic, since it requires a low pH (around 5.4–4.6) to grow effectively. The bacterium has
complex nutritional requirements. (B) Lactic acid production from whey by Lactobacillus
bulgaricus.
Source: (A)http://mrrohanbio.wikispaces.com/Lactobacillus+Bedlbrueckii
(B) Source: Author, ILLL in house
 Apart from fermenting milk, LABs are also used to ferment vegetables, meat, fish and
cereals. These can also improve taste and texture of fermented food. Finally, LABs play an
important role in the production of alcoholic beverages.
 In case of homofermentative bacteria (Lactobacillus sp.), very little substrate is used for
producing cell mass and other metabolites and majority of the carbon source is converted to
lactic acid, and here the percent conversion of sugars to lactic acid is virtually equivalent to
the theoretical yield of two moles of lactic acid per mole of hexose sugar utilized.
Production of Lactic acid
 LABs are categorized into two groups: i) Homolactic fermenters which utilizes the glycolytic
pathway and directly reduce almost all their pyruvate to lactate with the enzyme lactate
dehydrogenase; and ii) Heterolactic fermenters produce substantial amounts of products other
than lactate for example ethanol and CO2 by way of the phosphoketolase pathway. Commercial
production of lactate utilizes only homolactic fermenting strains of LABs
 In both, the starter culture is maintained in skimmed milk.
 The typical medium employed in the fermentation process consists of 10-15 % dextrose, 10 %
calcium carbonate, and small amounts of nitrogenous substances such as malt sprouts and
(NH4)2HPO4(0.25 %). LABs have complex nutritional requirement hence medium is
supplemented with B vitamins and some amino acids. Incubation temperature is 43-45C for
48 hours.
 The free lactic acid produced must be neutralized as it is toxic to the bacteria. The pH of the
fermenting medium is maintained between 5.5 and 6.5 by addition of calcium carbonate or
calcium hydroxide. This precipitates the free lactic acid by forming calcium lactate.
 Since lactic acid fermentation is an anaerobic process no aeration of the broth is required but
agitation is essential for proper mixing the calcium hydroxide to react with lactic acid
produced.

Figure 29: Biosynthesis of Lactic acid. Anaerobic fermentation of glucose produces lactic
acid in which pyruvate is reduced to lactic acid by lactate dehydrogenase and generating NAD
from NADH2 for glycolysis. The LABs produce either D(-)-lactic acid, L(+)-lactic acid or the
racemic mixture of both D and L isomers.
Recovery of Lactic acid
 After 48 - 72 hours of fermentation, contents of fermenter are boiled to coagulate protein.

 The filtrate containing calcium lactate is concentrated by removal of water under vacuum
followed by purification.
 A standard procedure for recovery from rather pure nutrient media is given in Figure 30.
 Broths from the fermentation of lower quality raw materials require even more extensive
purification steps, including pre-purification by filtering the hot calcium lactate solution, and its
repeated re-crystallisation.
 Alternatives used are solvent extraction (e.g. using isopropyl ether, 2-butanol or tri alkyl
tertiary amines in organic solvents), or esterification with methanol and subsequent
distillation.
 Ammonia can be used as an alternative to calcium bicarbonate and lactic acid can be
recovered by esterification. But this increases the cost of fermentation considerably.

Figure 30: With the addition of lime or chalk, the raw materials are
fermented in a fermenter and crude calcium lactate is formed. Calcium is
removed as calcium sulphate (gypsum) by adding sulphuric acid to the crude concentrated
calcium lactate resulting in crude lactic acid. The crude lactic acid is further purified and
concentrated to get L (+) lactic acid.

Summary
 Industrial fermentation can be applied for the production of i) Biomass; ii) Extracellular
metabolites iii) Enzymes and other proteins; and iv) Substrate transformations.
 Several of the metabolites produced by yeast, fungi and bacteria are useful to us food
supplements & alternative; cosmetic ingredients; antibiotics and as input to industry for
producing a variety of commercially important products.
 Industrially important strains have been derived from prototrophic strains by classic
technique of medium replacement or through genetic manipulations to maximise production
yields.
 Primary metabolites are produced during active period of growth and are essential for growth
and reproduction.
 Secondary metabolites are produced in response to specific environmental conditions and
during stationery phase of growth.
Primary metabolites Secondary metabolites

Amino acids Antibiotics
Vitamins Pigments
Nucleic acid Toxins
Polysachchrides Alkaloids
Ethanol Steroids
Lactic acid Polymeric substances eg gums, rubber
 Large scale fermentation purposes are specifically adjusted to microbial growth conditions.
 Downstream processing i.e., recover, purification, packaging and shipment are of equal
significance.
 For production of penicillin, a submerged fermentation process is followed with high aeration
and agitation with 25-27ºC with pH maintained at around 5.5-6.0. Lyophilized spores of
Penicillium are used as inoculum.
 The industrial production of streptomycin is carried out using submerged fermentation
processes. Streptomyces griseus is the principal strain for industrial production. The
fermentation is carried out at 28-30ºC with pH maintained at 7.6-8. The fermentation lasts
for 5-7 days with of yield of 1-3 g/L of the fermentation broth.
 L-Lysine can be produced in a two-step process by combination of Lysine-histidine double
auxotrophic mutant Escherichia coli and Aerobacter aerogenes. Industrial method utilizes one
step single strain approach majorly relying on Corynebacterium glutamicum.
 An important feature of industrial production of L-glutamic acid (monosodium glutamate,
MSG) using Corynebacterium glutamicum is permeabilization of rigid cell wall which otherwise
is an intracellular aggregate.

 Lactic acid producing bacteria or LABs is used to ferment milk, vegetables, alcoholic beverages
meat, fish and cereals. These can also improve taste and texture of fermented food.
Exercise/ Practice
1. Define the following:

a. Auxotrophs
b. Idiolites
c. Homofermentative LAB
d. Biosynthetic penicillin.
2. How are industrial microorganisms different from microorganisms in nature? In what

ways are they similar?
3. List three examples of primary metabolites and give their source organism.
4. Compare and contrast the production of natural, biosynthetic, and semi-synthetic

penicillins.
5. What are the factors that influence production of secondary metabolites?
6. Explain how can Corynebacterium glutamicum be used for production of L-Glutamic acid
and L-Lysine.
7. Explain different methods for the production of vinegar.
8. Write a short on LABs.
9. How are antibiotic producing strains isolated and screened.

Glossary
 Fermentation: Anaerobic catabolism of an organic compound in which the compound serves

as both an electron donor and an electron acceptor and in which ATP is usually produced by
substrate-level phosphorylation.
 Metabolome: The total complement of small molecules and metabolic intermediates of a cell
or organism.
 β-Lactam: Antibiotic a member of a group of antibiotics including penicillin that contain the
four-membered heterocyclic β-lactam ring.
 Broad-spectrum antibiotic: An antimicrobial drug useful in treating a wide variety of
bacterial diseases caused by both gram-negative and gram-positive bacteria.
 Industrial microbiology: Large-scale use of microorganisms to make products of
commercial value.
 LAB: Lactic acid producing bacteria.

References/ Bibliography/ Further Reading

1. Brock Biology of Microorganisms (13th Edition) by: Michael T. Madigan, John M. Martinko,
David Stahl, David P. Clark. Benjamin Cummings.
2. Microbiology: Principles and Explorations (8th Edition) by: Jacquelyn G. Black. John Wiley &
sons, Inc.
3. Crueger and A. Crueger; Biotechnology: A Textbook of Industrial Microbiology (Eng. Ed. T. D.
Brook). Sinaeur Associates, 1990.
4. L. E. Casida, Jr.; Industrial Microbiology. Wiley Eastern Ltd.
Suggested Readings
1. Derkx PM, Janzen T, Sørensen KI, Christensen JE, Stuer-Lauridsen B and Johansen E. The art
of strain improvement of industrial lactic acid bacteria without the use of recombinant DNA
technology. Microbial Cell Factories 2014, 13(Suppl. 1):S5 doi:10.1186/1475-2859-13-S1-
S5
Further Readings
1. Garg N, Jit S. 2015. Fermentation Process.
https://drive.google.com/file/d/0B0Izh6GcIA_DV2ViRmJnNW5seXM/view?pli=1.
2. Garg N, Jit S. 2015. Fermentation Products and
DownstreamProcessinghttps://drive.google.com/file/d/0B0Izh6GcIA_DMVdfWVBBMzNUVDA/v
iew?pli=1.
Useful Web Links

https://www.mdpi.com/1422-0067/13/5/5482/xml
http://www.sciencedirect.com/science/article/pii/S1687850714000314


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Applications of microbes in industry: Production of

primary and secondary metabolites

Lesson: Applications of microbes in industry:

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Figure 1: Production of primary and secondary metbolites.

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 Involved in growth, development and reproduction. Hence, essential

Figure 2: Over production of primary metabolite. To maximize

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Figure 4: Scheme for industrial production of metabolites.

Industrial Production of Microbial Metabolites

1. Screening, selection, maintenance of source microorganism for the production of target

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3. Sterilization of the medium, fermenter and ancillary equipment.

6. The extraction of the product and its purification.

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Figure 5: Structure and Function relationship of Penicillin:

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Value addition: Did you Know?

Heading text: Action of penicillin

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Diagram depicting formation of cross-links in the bacterial cell wall by a penicillin

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Value addition: Did you Know?

 In general, commercial penicillin is produced by fed-batch fermentation process using

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Figure 6: Summary of penicillin production and recovery.

 The media is placed at 25-27ºC with pH maintained at around 5.5-6.0.

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Figure 8: Derivation of natural penicillin to produce semisynthetic and

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Figure 9: Analogs of Penicillin.

 Streptomycin is a broad spectrum antibiotic (antimycobacterial) belonging to oligosaccharide

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Figure 10: Isolation and screening of antibiotic producers.

Figure 11: Structure of Streptomycin.

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 Streptomycin is derived from the bacterium Streptomyces griseus.Streptomycin has three

Figure 12: Streptomycin production by S. griseus. Glucose and other

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Figure 13: Phases of streptomycin production.

 On completion of fermentation, the mycelium is separated from the broth by filtration.

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Production of amino acids

Figure 14: Structure of Lysine. Abbreviated as Lys or K, is an α-amino acid. Lysine's

 Lysine is produced in a two-step process by combination of two bacterial strains:

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Figure 15: Biotransformation of Diaminopimelic acid (DAP) to lysine by

 Another industrial method utilizes one step single strain approach.

Figure 16: Single step production of lysine using mutant

 To achieve higher production values mutants of C. glutamicum are used in which

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 In such mutants, feedback inhibition of this enzyme by lysine is nearly eliminated.

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Figure 17: Enzymes involved in the biosysnthesis of L-glutamate

Production of Glutamic acid

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Recovery of Glutamic acid

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Figure 18: Biosynthetic production of Glutamic acid production by