Exopolysaccharides of Extremophiles

1. Exopolysaccharides of
Extremophiles
Dr.J.Kanimozhi
Associate Professor
Department of Biotechnology
Kalasalingam Academy of Research and Education
Indo-US Workshop on EXTREMOPHILES IN BIOTECHNOLOGY

2. Extremophile (“extreme-lovers”)
• Extremophile: a microorganism, especially an archaean, that lives
in conditions of extreme temperature, acidity, alkalinity, or
chemical concentration.
• Extremophiles include members of all three domains of life, i.e.,
bacteria, archaea, and eukarya. Most extremophiles are
microorganisms (and a high proportion of these are archaea), but
this group also includes eukaryotes such as protists (e.g., algae,
fungi and protozoa) and multicellular organisms.
Extremophilic and
Extremotolerant

3. Extremophiles and Extreme Environments
Psychrophiles
(extreme cold)
Organisms with the ability to survive at temperatures
below -4° F.
Thermophiles
(extreme heat)
Organisms with the ability to survive at temperatures of
140° F or even higher.
Radioresistant Microbes
(extreme radiation)
Organisms that can consistently survive doses of radiation
that are 500 times greater than the lethal dose for humans.
Alkaliphiles
(extreme bases; high pH levels)
Organisms with the ability to survive and thrive in
substances capable of neutralizing strong acids
(environments with pH values ranging from 9 to 11).
Acidophiles
(extreme acids; low pH levels)
Organisms that survive in highly acidic environments
(where the pH value rarely rises about 2).
Halophiles
(extreme saltiness)
Organisms that can survive in extremely salty environments
(5 to 10 times saltier than ocean water).
Xerophiles
(extreme dryness; lack of water)
Organisms that can grow and reproduce in conditions with
very little water available.
Barophiles
(extreme pressure)
Organisms that live in highly pressurized environments,
such as the bottom of the ocean.
Endoliths
(extreme rockiness)
Organisms with the ability to survive within solid rock, or
deep within the Earth’s crust.

4. Extremophiles- The research is still very preliminary
• 1991 to 2019
• Totally 571 articles are available related
to extremophile (Pubmed)
• 50% of the articles were reported from
2014 (297 articles from 2014-2019)
• //doi.org/10.1016/j.carbpol.2018.10.011
Biology of Archaea

5. Scientist Interest on Extremophiles
0
200
400
600
800
1000
1200
267
1116
21
134
234
512
14 18
NumberofPublishedArticles
Extremophiles
https://www.ncbi.nlm.nih.gov/pubmed/ (as on Nov 2019)
Geobacillus thermodenitrificans (116)
Thermus aquaticus (652)
Caldicellulosiruptor (248)

6. Key Features about Thermophilic EPS
 Short fermentation processes (often lasting several hours) due to the high growth
rate at elevated temperature and lower concentration of nutrient components .
 Good mass transfer at high temperature for cultivation
 Viscosity of culture liquid is lower at high temperature that suggests lower energy
consumption
 Performance of processes at high temperature reduces the risk of contamination
 Non pathogenic products from thermophiles are applicable in food and cosmetic
industry EPSs synthesized by thermophilic bacteria and archaea are suggested to
keep their emulsifying and rheological properties at high temperature, in which
many processes in food industry are performed.
 Usually thermostable molecules can remain effective even at extreme conditions
 of pH, temperature, and salinity due to their more rigid molecule.
 They form stable oil/water emulsions needed for cosmetic industry

7. Thermophiles in biotechnology
DNA polymerases
For PCR based technologies
Thermophiles (Thermus aquaticus,
Pyrococcus furiosus, and Thermococcus
litoralis)
Biofuel production
Thermophile-Thermoanaerobacterium
saccharolyticum utilize hemicellulose and
pentose sugars like xylose
Acidophiles and Halophiles
Extremozymes
Protease & Lipase
Psychrophilic proteases
to enhance cold water washing
lipases from thermophilic Bacillus
Biomining
(bioleaching-removal of insoluble metal
sulfides or oxides by using microorganisms)
Acidophiles
thermophiles
Extremophilesin
biotechnology

8. Extremophiles: a Promising
Source of Exopolysaccharides
with Interesting Properties

9. MICROBIAL EXOPOLYSACCHARIDES
Polysaccharides are high molecular weight carbohydrate polymers composed of long
chains of monosaccharide units bound together by glycosidic linkages.
They are natural, non-toxic, and biodegradable polymers that cover the surface of most
cells and play important roles in various biological mechanisms such as immune response,
adhesion, infection, and signal transduction.
Sources
•Algae,
•Higher order plants,
•Microbial - bacterial or fungal cultures.
Advantages of Microbial Polysaccharide
Enables fast & high yielding production
Process conditions can be fully controlled
Geographical or seasonal variations do not affect the process
Short duration
Energy efficient, in the case of microalgae (production uses solar energy);
Possibility of utilizing agro industrial wastes
Pretreatment Techniques for Bio fuels and Bio refineries- Green Energy and
Technology Microbial Production of Extracellular Polysaccharides from Biomass by
Ebru Toksoy Öner

10. Possible physiological roles of EPS in microbial
cells that could contribute surviving in extreme
conditions
Role of EPS in microbial cells
Physical
Barrier against
abiotic factors
Prevention of
entering harmful
substances
Binding and
neutralization
Contribution
in providing
 Temperature
 pH
 Pressure
 Radiations
 Toxic metals
 Toxic compounds
 Bacteriophage
 Protozoans
 Nutrient
 Water
 Metal ions
DOI: 10.1007/978-3-319-13521-2_4

11. EPS-(Extracellular Polymeric Substances/Extracellular
Polysaccharides)
Aeribacillus pallidus
Weissella cibaria
Xanthomonas compestris

12. BACTERIAL EXOPOLYSACCHARIDE (EPS)
EPS are often favored due to
• Naturally exuded by most microorganisms into the extracellular
environment.
• Facilitating the easier recovery.
• Structural diversity and peculiar characteristics.
• In contemporary, EPSs are potential applicants for many industrial
uses especially in the food and pharmaceutical industries.
• Among many microbial EPS, Polysaccharides are often preferred
for the human use due to ease of production and recovery and also
it has the low antigenicity.
05.04.17 12

13. Monomers of EPS of Extremophiles
• arabinose (Ara), ribose (Rib), xylose (Xyl)
• glucose (Glc), galactose (Gal), mannose (Man)
• quinovose (Qui), fucose (Fuc), rhamnose (Rha)
• glucuronic acid (GlcA), galacturonic acid (GalA), mannuronic acid (ManA)
• glucosamine (GlcN), galactosamine (GalN), mannosamine (ManN)
• 3-deoxy-D-manno-2-octulosonic acid (Kdo), neuraminic acid (Neu)
Extremophiles (Thermophiles) mostly produces
Heteropolysaccharides
Psychrophiles produces Homopolysaccharides

14. EPSs Produced by Archaeal Species

15. Potential Application of Extremophile EPS
Extremophiles Properties Applications
Thermophiles
Thermostability
Immunoregulation
Emulsifying
Biomaterial
Biomedicine
Food Industry
Psychrophiles
Cryoprotection
Heavy Metal Binding
Emulsifying
Biomaterial
Food Industry
Halophiles
Emulsifying
Antioxidancy
Pseudoplasticity
Heavy metal binding
Antitumor Activity
Biomaterial
Biomedicine
Food Industry
Acidophiles Heavy Metal Binding Bioleaching
Alkaliphiles
Emulsifying
Pseudoplasticity
Food Industry

16. Production and Characterisation of EPS of Extremophiles and their
applications.
Phase-I Choice
of Material
Isolation and Identification
of EPS/CPS producing
extremophiles from extreme
conditions
Characterisation of isolate
and EPS produced (simple
Tests) and potential
Applications
Optimization
(Laboratory
Scale)-Batch
Process
Detailed Structural
Elucidation and
Exploration of EPS
Phase-II (thermophilic
Bioprocessing)
Yield Maximization
Phase-III
Microbial Fermentation
Response Surface
Methodology-Genetic
Algorithm (RSM-GA)
based technology.
Alternate Production
strategies (Reactor
Choices)
Correlation between yield
and extreme conditions
GENERAL VIEW OF RESEARCH
16

17. General scheme illustrating detection,
extraction and purification of
exopolysaccharides

18. -cd

19. General strategies for the production of
bacterial exopolysaccharide
Fermentation
• Microbial
• Enzymatic
Conversion
Extraction
• Cell removal
• Centrifugation
• Filtration
• Polymer
precipitation
• Methanol
• Ethanol
• Isopropanol
Purification
• Reprecipitation
• Ethanol
• Methanol
• Isopropanol
• Deproteinization
• Salting out
• Protease
• Ultrafiltration
Drying
• Freeze Drying
• Lyophilization
• Drum Drying
Characterization
• Chemical
Structure
• Monomer analysis
• GC-MS, FTIR,
NMR
• Size exclusion
chromatography
Characterisation of polysaccharides primary and secondary
structures performed during each step of purification
Acid/Enzymatic Hydrolysis
Monomer Analysis by GPC
FTIR, TLC, Biochemical
Assay, PAGE Analysis, Size
Exclusion
chromatography, SEM

20. EPS of Psychrophiles Applied in
CryopreservationVaccines Organ Preservation
Human cell isolation kits* Frozen and Dried Foods Ice creams

21. Psychrotolerant bacterium produces
exopolysaccharide (EPS) at low temperature
https://doi.org/10.1016/j.intimp.2018.04.039

22. Molecular weight distribution and Thermo
gravimetric analysis of EPS

23. Xerophiles: Tolerating Extreme Desiccation
• Many mold and yeast species are
xerophilic. Mold growth on bread is an
example of food spoilage by xerophilic
organisms. It includes,
• Trichosporonoides nigrescens,
• cacti, and
• tardigrades,
• which can survive for almost a decade
without exposure to water.
• Cyanobacteria are known to produce
both compatible solutes at intracellular
level and a copious amount of
exopolysaccharides as a protective
coat. Gloeocapsopsis sp.UTEX B3054
Transmission electron
photomicrograph Gloeocapsopsis sp.
UTEX B3054
multilayered and
abundant EPS
Aspergillus
candidus

24. EPS of Halophile
Transmission electronic microscopy
photograph of S. mucosus strain A3T stained
with the specific stain for polysaccharide
ruthenium red. Bar: 1 μm. Arrow indicates the
EPS of the strain.
Mar. Drugs 2010, 8, 2240-2251; doi:10.3390/md8082240
Excellent emulsification activity
with vegetable oils
Salipiger mucosus A3T excretes
significant quantities of EPS when
cultivated under optimum growth
conditions and possess sulfate in
EPS
Sulfated EPSs are of great potential interest
in medicine since they have a number of
bioactive properties: anticoagulant,
antiangiogenic, antiproliferative, antiviral,
etc.

25. Functional groups of EPS of Halophile
• Phosphate groups, which have also been
observed in other EPSs , could confer
important properties on them because
they are essential to the activation of
lymphocytes and in some antitumoral
processes.
• Fucose and fucose-rich oligosaccharides
can be used in biocosmetics, in the field
of medicine and in the food industry.
• The polymer from S. mucosus strain A3T
may prove to be a simple source of
fucose, as reported for the EPSs excreted
by Klebsiella pneumoniae and
Clavibacter michiganensis. Halophiles are excellent
bioflocculant-producers
Halobacterium salinarium,
Haloferax volcanii,
Halobacterium distributum
Schematic of flocculation mechanisms; a) charge
neutralization, b) bridging, c) electrostatic patch, d)
sweeping (Vandamme, 2013)
Azo dyes decolorization under high alkalinity
and salinity conditions by Halomonas sp. Is
also reported

26. Role of EPS on metal Biosorption
Diagrammatic representation of hydrophilic anionic
EPS of bacterial cell containing carbonyl (CO),
phosphate(PO), cyanide(CN), hydroxyl (eOH) and
amino (eNH) groups that bind to cationic lead(Pb)

27. Prospective of Microbial Exopolysaccharide for
Heavy Metal Exclusion
https://link.springer.com/article/10.1007/s12010-017-2591-4

28. Role EPS on Metal Sorption
• ADVANTAGES
• Environmentally safe and cost
effective adsorbent
• No generation of toxic byproducts
• Both live and dead cell bound EPS can
be used for metal adsorption, dead
cells are devoid of hazards posed by
living cells
• Requires milder operating conditions
compared to conventional
physiochemical methods
• Sensitive and can carry out
sequestration even at lower metal ion
concentrations
• Can be recycled after desorption of
adsorbed metal ions and the metal ions
recovered can be further exploited
Hyphomonas MHS-3, Hyphomonas sp. The
marine strains were able to remove the metal
ions from an initial concentration of 50–100
ppb to US EPAa drinking water standards
WO1998030503 A1

29. Thermophiles as a Promising Source of
Exopolysaccharides
Thermophilic EPS producers were isolated from
both, Bacteria (Thermotoga, Thermus, Bacillus Geobacillus,
Brevibacillus, Aeribacillus) and Archaea (Thermococcus, Sulfolobus)
domains.
Advantages in using thermophilic processes for EPS production
 high molecular weight,
 stability of their molecules,
 good synergism with other hydrocolloids,
 biological activity against cytotoxic compounds,
 antiviral and immunostimulating activities
These attributes determine their possible future applications.

30. Bacillus and Geobacillus
• Arena et al. reported the
immunomodulatory and
antiviral effects of an EPS
produced by a strain of
Geobacillus
thermodenitrificans, isolated
from a shallow marine vent of
Vulcano island, in Italy.
• The EPS showed a molecular
weight of 400 KDa and
displayed mannose and
glucose as main
monosaccharidic components.
Geobacillus stearothermophilus
(previously Bacillus
stearothermophilus) is a rod-shaped,
Gram-positive bacterium and a
member of the division Firmicutes.
The bacterium is a thermophile and is
widely distributed in soil, hot springs,
ocean sediment, and is a cause of
spoilage in food products.

31. Biotechnological Applications of Marine Bacterial
EPS
• Apart from EPS, extremophiles are known to generate
antimicrobial peptides and diketopiperazines
• Antimicrobial peptides have been found in the
Halobacteriaceae (phylogenetic family containing all
halophilic archaea) as well as Sulfolobus species.
• These peptides (halocins) from halophilic archaea are
thought to be found in all species of the family. Each halocin
has a specific range of activity, and some act on a broader
range of microorganisms than others.
• Halocins have been shown to be effective at killing archaeal
cells; however, there are no data to show that halocins kill
microorganisms pathogenic to humans. Interestingly, there is
• evidence that they assist canines in recovering from surgery

32. Thank You
05.04.17 32