Recent advances in antibiotics, vaccine, biocide production and steroid production

1. Presented by
Guruprasad A
PAMB 1083
Current advances in production of
Antibiotics , Vaccines, Biocides and
Steroid transformation
PBT 603 (1+1)
Advances in Microbial Biotechnology

2. Advances in Antibiotics
production

3. Antibiotics
•Compound that kill or inhibit the growth of other organis
•Most Antibiotics are produced by filamentous fungi or A
•They are derived from special microorganisms or
other living systems, and are produced on an industrial
scale using a fermentation process.
• Today, over 10,000 antibiotic substances have been repo

4. Properties of Microorganisms required for
industrial production
Grow rapidly on large scale in inexpensive medium.
Produce desired product quickly.
Should be Non-pathogenic.
Amenable for genetic modification

5. Production of Antibiotics
• Production of Antibiotics began during second world war with
the production of Penicillin
• Now most antibiotics are produced by staged fermentation, in
which strains of microorganisms producing high yields in
optimum conditions are used

6. Production techniques
❑ Production of antibiotics can be done by 3 methods.
❑ 1. Natural microbial production using Fermentation technology.
Example: Penicillin
❑ 2. Semi synthetic production (post production modification of natural
antibiotics).
Example: Ampicillin
❑ 3. Synthetic production of antibiotics made synthetically in the lab.
Example: Quinoline

7. Fermentation technology
o The source microorganism is grown in large containers
(100,000-150,000 litre or more) containing a liquid growth
medium.
o Oxygen concentration, temperature, pH and nutrient levels
must be optimum.
o As antibiotics are secondary metabolites, the population size
must be controlled very carefully to ensure that maximum yield is
obtained before the cells die.

8. Requirement
The fermentation process requires the following
1. A pure culture of the chosen organism, in sufficient quantity.
2. Sterilized, carefully composed medium for growth of the organism
3. A seed fermenter, a mini-model of production fermenter to
develop inoculums to initiate the process in the main fermenter.
4. A production fermenter, the functional large model and
5. Equipment for: a) Drawing the culture medium in steady state b)
Cell separation c) Collection of cell free supernatant d)
Product purification e) Effluent treatment.

9. Major techniques (Genetic engineering)
Species are often genetically modified to yield maximum amounts of antibiotics.
o Mutation is often used -introducing mutagens such as
ultraviolet radiation, x-rays
o Selection and further reproduction of the higher yielding
strains can raise yields by 20-fold or more.
o Another technique used to increase yields is gene
amplification, where copies of genes coding for enzymes
involved in the antibiotic production can be inserted back into
a cell, via vectors such as plasmids.

10. Raw materials
o The compounds that make the fermentation broth are the primary
raw materials required for antibiotic production.
The broth is an aqueous solution made up of all of the ingredients
necessary for the proliferation of the microorganisms.
• Typically, it contains;
o Carbon source: molasses, or soy meal, acetic acid, alcohols, or
hydrocarbons
• These materials are needed as a food source for the organisms.
o Nitrogen source: Typically ammonia salt is used

11. Other elements
o Trace elements needed for proper growth of antibiotic
producing microorganisms such as:
• Phosphorus • Sulfur • Magnesium • Zinc.
o Anti foaming agents to prevent foaming during fermentation
such as: • Lard oil • Octadecanol

12. Steps in production
• First the organism that makes the antibiotic must be identified.
• Desired microorganism must then be isolated.
• Then the organism must be grown on a scale large enough to
allow the purification and chemical analysis of the antibiotic.
• The antibiotic tested against a wide variety of bacterial
species.
• It is important that sterile conditions be maintained throughout
the manufacturing process, because contamination by foreign
microbes will ruin the fermentation.

13. A) Seed culture/ Starting culture
• Before the fermentation process the desired microbe must
be isolated and its number must be increased by many
times.
• A starter culture from a sample is usually created in the lab
• First the sample organism is cultured in agar plate
• This will be transferred into a shake flask containing
necessary nutrients for growth
• Later the suspension will be transferred into the seed tanks

14. Diagrammatic representation

15. Seed Tank
Operation
• The seed tanks are steel tanks designed to provide an
ideal environment for growing microorganisms.
• The seed tanks are equipped with mixers, which mix the
growth medium with microbes, and a pump to deliver
sterilized, filtered air.
• After about 24-28 hours, the material in the seed tanks is
transferred to the primary fermentation tank.

16. B) Fermentation
Fermentor
o The fermentation tank is a larger version of the seed tank, which is able
to hold about 1.5 lakh litres.
o Microorganisms are allowed to grow and multiply.
• During this process, they excrete large quantities of the desired
antibiotic.
• The tanks are cooled to keep the temperature between (23-27.2 °C).
• It is constantly agitated, and a continuous stream of sterilized air is
pumped into it.
o Anti- foaming agents are periodically added.
• Since pH control is vital for optimal growth, acids or bases are added to
the tank as necessary.

17. C) Isolation
Downstream process
After 3-5days, the maximum amount of antibiotic will have
been produced.
• The isolation process can begin.
• The isolation depend on the specific antibiotic produced,
the fermentation broth is processed by various purification
methods.

18. Water soluble antibiotics
• For antibiotic compounds that are water soluble, an ion-
exchange method is used for purification.
• The compound is first separated from the waste organic
materials in the broth.
• Then sent through equipment, which separates the other
water-soluble compounds from the desired one.

19. Oil soluble antibiotics
Solvent extraction
❑ Solvent extraction method is used for the isolation of oil soluble or
organic antibiotics.
o The broth is treated with organic solvents such as butyl acetate or
methyl isobutyl ketone, which can dissolve the antibiotic.
o The dissolved antibiotic is then recovered using various organic
chemical means.
o At the end of this step a purified powdered form of the antibiotic is
obtained which can be further refined into different product types.

20. D) Refining and packing
❑ Antibiotic products can take on many different forms. They can be
sold in solutions for intravenous bags or syringes, in pill or gel capsule
form, or powders, which are incorporated into topical ointments.
❑ Various refining steps may be taken after the initial isolation.
• For intravenous bags, the crystalline antibiotic can be dissolved in a
solution, put in the bag, which is then hermetically sealed.
• For gel capsules, the powdered antibiotic is physically filled into the
bottom half of a capsule then the top half is mechanically put in place.
• When used in topical ointments, the antibiotic is mixed into the
ointment

22. Quality control
o Quality control is of great importance in the production of antibiotics. •
Since it involves a fermentation process, steps must be taken to ensure
that absolutely no contamination is introduced at any point during antibiotic
production.
o During manufacturing, the quality of all the compounds is checked on a
regular basis.
• Frequent checks of the condition of the microorganism culture during
fermentation.
• Various physical and chemical properties of the finished product are
checked such as pH, melting point, and moisture content.

28. Antibiotics
Disadvantages

29. Antibiotics
Fading Miracle ?

33. Advances in Vaccine production

34. Vaccine ?

41. Basic steps in Vaccine production

42. Selection of strains for Vaccine production
Growing the Micro organisms
Isolation and Purification
Inactivation or Attenuation
Formulation of Vaccine
Quality control and Lot release

44. https://www.youtube.com/watch?v=2t_mQwTY4WQ

45. Current advances
What we are doing ?
•Use of recombinant DNA technique to insert the gene coding for the
protein of interest into the genome of avirulent virus that can be
administered as vaccine
•Including in the vaccine only those subviral components needed to
stimulate protective antibody, minimizing occurrence of adverse reactions
•Use of purified proteins isolated from purified virus or synthesized from
cloned genes (recombinant Hep B vaccine containing viral proteins
synthesized in yeast cells)- forming empty VLP •Use of synthetic peptides
corresponding to antigenic determinants on a viral protein, thus avoiding
reversion to virulence since no viral nucleic add is present (newer HIV
vaccines)

46. Current advances
What we are doing ?
•Development of edible vaccines where transgenic plants synthesizing
antigens from pathogenic viruses provide new cost effective way of vaccine
delivery
•Use of naked DNA vaccines in which recombinant plasmids carrying the gene
for the protein of interest are injected into hosts and the DNA produces
immunizing protein
•Administration of vaccines locally to stimulate antibody at the portal of entry
(aerosol vaccines for respiratory disease viruses)
•Needle free adminstration: oral (Live Bacterial Vector, Particulate
Formulations, Mucosal Adjuvants) transcutaneous, liquid jet injection and
epidermal powder immunization, microneedle-based injection system

47. Reverse Vaccinology
Objectives
• • To minimize the Laboratory based research.
• • To develop a computational analysis of Antigens using Bioinformatics
tools.
• • To design a molecule that can replace an antigen in detection process.
• • For the development of immunodiagnostic tests and vaccines.
• • For detection of antibodies produced as a result of infections, allergies,
autoimmune diseases, or cancers.

48. Reverse Vaccinology ?
What it is ?
• The technique of identifying the proteins that are exposed on
the surface by using genome instead of the microorganism,
this novel approach is known as "reverse vaccinology"
• Reverse vaccinology is an improvement on vaccinology that
employs bioinformatics, pioneered by Rino Rappuoli and first
used against Serogroup B. meningococcus.

51. Available softwares
For epitope prediction
Computer Aided bioinformatics projects are extremely popular, as they
help guide the laboratory experiments. Some of them popularly used for
Reverse Vaccinology are
• NERVE - one relatively new data processing program
• Vaxign- an even more comprehensive program, was created in 2008.
Vaxign is web-based and completely public-access extremely accurate
and efficient
• RANKPEP — an Online software, for the peptide bonding predictions.

54. DNA Vaccines
A promising future
•DNA vaccines are third generation vaccines, and are made up of
a small, circular piece of bacterial DNA (called a plasmid) that
has been genetically engineered to produce one or two specific
proteins (antigens) from a micro-organism. The vaccine DNA is
injected into the cells of the body, where the "inner machinery" of
the host cells "reads" the DNA and converts it into pathogenic
proteins

55. DNA Vaccines
Advantages
•Cheaper and easier to produce
•Large rapid GMP manufacturing capabilities
•No need to handle infectious pathogens during production
•Safer
•Can elicit both humoral & cell mediated immunity
•Stable at a broad range of temperature no cold-chain requirement)
•Can be designed and produced by genetic engineering to have only the desired
antigens or antigenic sequences (epitopes) in the vaccine
•Ability to immunize against multiple antigens and/or Pathogens
•Nonviral and no induction of anti-vector immunity

56. Future aspects to work on
Present day limitation
• Single disease prevention
• Require Multiple doses
• Not 100 % effective
• No sustained Protection
• Though less, but have adverse reactions
• Most are not safe in pregnancy and immunodeficiency
• Biological & environmental stability- difficult
• Cost effectiveness • Risk of infection with live-attenuated micro-organisms

57. Will I see one of these in my life ?

58. Ethical concern?

60. Combination vaccine?

63. Advances in Biocide production

64. Biocide ?

65. Biocide
Definition
• A biocide is basically a formulation containing one or
more active substances that will at very small doses
repel or control or destroys harmful organisms

66. Facts
About biocides
■ Over 3 mmt of biocides are used annually on Earth
■ Approximately 25% of the biocides used to houses,
gardens, lawns, parks, playing fields, swimming pools.
■ The average USA homeowner applies 2-6 times more
biocides per acre than farmers do!

67. Why we have not heard about biocides ?

68. Global Consumption of Biocides

71. Biocide V/S Pesticide

72. Classification of Biocides
There are two types of Major Biocides
1. Natural Biocides
2. Chemical Biocides e.g - Calcium Hypochlorite,
Hydrogen peroxide, Chlorine Beach etc….

73. Natural Biocides
Microbial Insecticides
Single cell organisms, such as bacteria, fungi and protozoa, and
viruses, have been mass produced and formulated for use in a
manner similar to insecticides.
• Microbial insecticides can be
1. Microbially produced toxic substance
2. Organism

74. Bacterial biocide
BT (Bacillus thuringiensis)

75. Actinomycetes biocide
Spinosad, Milbemectin, Avermectin

76. Viral biocide
Baculovirus (NPV, Granulosis virus)

77. Fungal biocide
Green Muscardine and White Muscardine

78. Fungal biocide
Green Muscardine and White Muscardine

81. Common Micro-organisms
Biofilm producing organisms
• One of the popular antimicrobial agents is surfactants, which are produced synthetically and
biologically. Biosurfactants (biologically derived surfactants) are the secondary metabolites and
surface-active amphiphilic compounds of biological origin, synthesized by specific bacteria, fungi,
and yeasts, with Pseudomonas aeruginosa, Bacillus subtilis, Candida albicans, and Acinetobacter
calcoaceticus as dominant species.
• The compounds can be secreted into the external environment, form part of the cell membrane,
or be metabolized within the cell .
• They are non-ribosomally synthesized compounds that display noticeable emulsification and
surface activities.
• Biosurfactants form a diverse group of biomolecules with molecular weights ranging from 500 Da
to 1000 kDa.
• Based on their chemical composition and microbial origin, biosurfactants have been classified into
different groups. There are five major classes: glycolipids, lipopeptides, phospholipids, polymeric
compounds, and neutral lipids
• These compounds are known to exhibit broad-spectrum antimicrobial activity, and different
classes of biosurfactants are being used by the agricultural, oil, food, cosmetic, biotechnological,
and pharmaceutical industries, as well as in a wide range of environmental remediation
technologies.

83. Advantages
Over synthetic Biocides
• Biosurfactants also destroy microbial cells by directly disrupting the integrity of the
plasma membrane or cell wall.
• The magnitude of such damage to the cell boundary makes it difficult for any target
organism to develop resistance to the biosurfactant
• For example, lipopeptides create pores in the cell membrane of the target organism,
creating an imbalance in the movement of ions both into and out of the microbial cell
that is lethal to the damaged cell
• Glycolipid-based biosurfactants such as rhamnolipids primarily produced by
Pseudomonas species also display algicidal, anti-amoebal, and zoosporicidal
properties.
• Bacillus species are able to form biofilms and efficiently secrete a wide range of
antimicrobial compounds, such as polymyxin B, gramicidin S, and biosurfactants,
which belong to the lipopeptides family. They seem to be promising candidates to
produce antimicrobials against sulphate-reducing bacteria (SRB).

84. Steroid transformation

85. Steroids
What are steroids ?
• Steroids are small organic molecules with a characteristic
molecular structure containing four rings of carbon atoms
synthesized in steroidogenic tissues
• It include many hormones, alkaloids, and vitamins
• It act on target sites to regulate a cascade of physiological
functions

86. Types of Steroids
• Sex hormones. These are the male hormones, including testosterone, which
together are called androgens, and the female hormones, including estradiol, a
type of estrogen.
• Corticosteroids. Hormones include cortisone and cortisol. They are thought to
have a role in the immune system.
• Mineralocorticoids. These hormones maintain the balance of sodium and
potassium in the body and include aldosterone.
• Bile salts or bile acid. These steroids are made in the liver. They don't function
as hormones, but are necessary for digestion and absorption of fats.
• Sterols. The most commonly known of these is cholesterol. Other sterols help
your body to make vitamin D from sunlight and to build cell walls.

87. Cholesterol Biosynthesis Pathway

88. Transformation of steroids
• Transformation of steroids means conversion of precursor
steroids to important drug intermediates and further
conversion of these intermediates to active compounds by
simple chemical or microbial processes.
• The chemical synthesis and transformations of steroids
requires multiple steps and makes the use of reagents that
have health risks and cause serious environmental disposal
problems.
• Alternative is microbial transformation

89. Microbial Transformation
Transformation of steroids
• These involves simple, chemically defined reactions catalyzed by enzymes
present in the cell.
• Microbial cells provide the enzymes to catalyze the transformation reactions.
• The microorganisms have got the ability to chemically modify a wide variety of
organic compounds. These microbes during The bioconversion provide
enzymes which act upon and convert the organic compound into other
compounds or modify it.
• Microbial transformations cleave the complex side chains of precursor steroids
in one single step and incorporate desirable modifications in steroid nucleus.
• Microbial transformations are regiospecific and stereospecific, whereby organic
compounds are modified into desirable isomers of products involving simple
chemically defined reactions catalyzed by the enzymes in the microbial cells.

90. Types of steroidal transformation
• Oxidation
• Hydroxylation
• Dehydrogenation
• Epoxidations
• Oxidation to ketone through hydroxylation
• Ring A Aromatization
• Degradation of steroid nucleus

91. Oxidation and Hydroxylation
Oxidation
• Oxidation of alcohols to ketone: 3β-0H to 3-keto
• Side chain cleavage of steroids
• Decarboxylation of acid
Hydroxylation
• Hydroxylation involves the substitution of hydroxyl group directly for the
hydrogen at the position, be it α or β, in the steroid with a retention of
configuration.
• The oxygen atom in the hydroxyl group is derived form molecular oxygen
(gaseous), not from water, and the hydroxyl group thus formed always retains
the stereochemical configuration of the hydrogen atom that has been replaced.

92. Fungi are the most active hydroxylating microorganisms, but same bacteria
particularly the Bacilli, Nocardia and Streptomyces show fair goad activity.

93. Dehydrogenation
• Dehydrogenation with the concomitant introduction of a double bond
has been reported for all four rings of the steroid nucleus
• The introduction of unsaturated bonds in Ring A is the only reactions
of commercial importance.
• Example : • In 1955, Chamey and co-worker observed that they could
greatly enhance the anti-inflamrnatory properties of cortisol by making
the compound to be dehydrogenated at first position by
Corynebacterium simplex.
• The resultant product, prednisolone, was 3-5 times more active than
the parent compound and produced fewer side effects.

94. Epoxidation
The epoxidation of steroidal double bonds are rare example of biological epoxidation

95. Ring A Aromatisation
• The microbial aromatization of suitable steroid substrates can
lead to ring A aromatic compounds, particularly the estrogens
which constitutes an important ingredient in oral contraceptives
drugs and play important role in replacement therapy for
menopause treatment
• Cell free extracts of Pseudomonas testosteroni could transform
19-nor-testosterone into estrone with small quantities of estradiol

96. Hydrolysis
Hydrolysis of esters
• Flavobacterium dehydrogenans contain a specific enzyme
acetolase which hydrolyses the steroidal acetates
•

97. Esterification
Usually involves acetylation

99. Thank you