Microorganisms produce two types of biopolymers to survive in extreme conditions: extracellular polysaccharides (EPSs) and endocellular polyhydroxyalkanoates (PHAs). EPSs are high molecular weight polymers biosynthesized by many microorganisms. They can be classified as homopolysaccharides or heteropolysaccharides depending on sugar composition. Microbes secrete EPSs for protective and adaptive functions. Commercial production involves optimizing fermentation conditions to improve yields for applications in pharmaceutical, food, and other industries.

1. Production of
Exopolysaccharides
Johnson Mwove
Jomo Kenyatta University of Agriculture and
Technology
Advances in Food Biotechnology and Genetic Engineering

2. Introduction
 There are two main types of biopolymers
produced by microorganisms that survive extreme
conditions. These are the:
 Extracellular polysaccharides (EPSs)
 Endocellular polyhydroxyalkanoates (PHAs)
 Exopolysaccharides (EPSs) are high molecular
weight and biodegradable polymers that are
biosynthesized by a wide range of microorganisms
(Madhuri & Vidya Prabhakar, 2014; Sanlibaba &
Çakmak, 2016).

3. Introduction
 EPSs can be classified into two groups.
 Homopolysaccharides are polymers which are
composed of one type of monosaccharide
 Heteropolysaccharides are polymers of
repeating units that are composed of two or
more types of monosaccharide
 Other organic or inorganic substituents can also be
found (Finore et al., 2014; Sanlibaba & Çakmak,
2016; Shukla, 2017).

4. EPS produced by Lactobacillus sp
 HoPS –
Homopolysa
ccharides
 HePS –
Heteropolysa
charides
 (Oleksy &
Klewicka,
2016).

5. Introduction
 Microbial EPSs generally exist in two forms
depending on their locations:
 Cell-bound EPSs which closely adhere to the
bacterial surface,
 Released EPSs that release into the
surrounding medium, as free EPSs.
 EPSs produced from LAB are distinguished as ropy
or non-ropy EPS (Sanlibaba & Çakmak, 2016)

6. Introduction
 (a) Encapsulated Lb. casei (×1000); (b) macroscopic
appearance of the “ropy” strand formed by the cellular
mass of a EPS-producing L. acidophilus growing on the
surface of de Man, Rogosa, and Sharpe (MRS) agar plates
(Oleksy & Klewicka, 2016).

7. Introduction
 EPSs are secreted by microorganisms for their survival
in harsh environmental conditions as a protective
mechanism (Poli et al., 2011; Shukla, 2017) rather than
as energy sources (Sanlibaba & Çakmak, 2016).
 EPSs are produced in response to biotic stress (e.g.,
competition), abiotic stress factors (e.g., changes
temperature, light intensity, pH, salinity) and/or as a
strategy of adaptation to an extreme environment like
in the case of acidophilic or thermophilic species
(Donot, Fontana, Baccou, & Schorr-Galindo, 2012;
Freitas, Torres, & Reis, 2017).

8. Selected functions of EPS in bacterial cells
 Adhesion
 Aggregation of bacterial cells and formation of
biofilms
 Protective barrier
 Sorption of exogenous organic compounds
 Sorption of inorganic ions
 Retention of water
 Nutrient source
 Interaction with enzymes
(Oleksy & Klewicka, 2016)

9. Examples of EPSs
 Dextran - Leuconostoc mesenteroides
subsp.mesenteroides and Leuconostoc
mesenteroides subsp.dextranicum
 Mutan - Streptococcus mutans and Streptococcus
sobrinus
 Alternan - Leuconostoc mesenteroides
 Reuteran - Lactobacillus reuteri
 Others include Levan, Inulin, Kefiran, Glucan etc.

10. EPSs biosynthesis
The four main steps of
the synthesis:
 Sugar transportation,
 Sugar nucleotide
synthesis,
 Repeating unit
synthesis, and
 Polymerization of the
repeating units formed
in the cytoplasm
(Donot et al., 2012;
Sanlibaba & Çakmak,
2016)
Intracellular
synthesis of the
polysaccharides
Exudation of
polysaccharides
out of the cell
Carbon
substrate
assimilation

11. EPSs biosynthesis
1. The sugar
transport
into the
cytoplasm
2. the
synthesis of
sugar-1-
phosphates
3.
activation
of and
coupling
of sugars
4. the
processes
involved in
the export of
the EPS

12. EPSs biosynthesis
 EPS production is
observed during all
growth phases but
increased during the
stationary phase, like
for Anabaena
flosaquae, Anabaena
cylindrical or
Botryococcus braunii.
 Inversely, Nostoc strains synthesise extracellular
polysaccharides in larger amounts in exponential
growth phase (Delattre et al., 2016).
0
1
2
3
4
5
6
7
0 5 10 15 20 25
LogMicrobialcounts
Time (Days)
Microbial growth

13. EPS Production
 Although the ability to secrete exopolysaccharides
(EPS) is widespread among microorganisms, only a
few bacterial (e.g. xanthan, levan, dextran) and
fungal (e.g. pullulan) EPS have reached full
commercialization.
 Other microbial EPS producers have been the
subject of extensive research, including
endophytes, extremophiles, microalgae and
Cyanobacteria, as well as mixed microbial
consortia. (Freitas et al., 2017).

14. EPS Producers
(Freitas et al., 2017)

15. EPS Production: Substrates
 Carbon availability concomitant with limiting nitrogen
is usually reported as favoring EPS production by
microorganisms.
 Under growth limiting conditions, the carbon source is
derived towards polysaccharide synthesis is (Delattre
et al., 2016; Freitas et al., 2017)
 Glucose and sucrose are the most commonly used
carbon sources for microbial cultivation and
production of EPS for most microorganisms.
 Other elements, such as phosphorus, potassium and
metal cations, are also required for microbial growth
and EPS synthesis.

16. Process operation conditions
 To assure a stable and reproducible bioprocess
performance, cultivation parameters, such as pH,
temperature, Dissolved Oxygen concentration,
irradiance, carbon dioxide supply and stirrer
speed
 These are often monitored and/or controlled
within defined ranges for different cultures.
 Mixing and aeration are also relevant parameters
as they determine the availability of nutrients and
oxygen (Freitas et al., 2017)

17. Cultivation mode
 At lab scale, small bioreactors with similar design as
those of the large scale production fermenters
 The selection of the most adequate cultivation mode
will depend on whether EPS production is growth
associated (e.g. gellan)or non-growth associated (e.g.
curdlan).
 Most microbial EPS production processes are simple
batch cultures or single pulse fed-batch cultures,
following exhaustion of nitrogen source in the medium
(Delattre et al., 2016; Freitas et al., 2017).
 Nevertheless, other cultivation modes are proposed,
including fed-batch and continuous culture.

18. Bioreactor design
 Stirred tank reactors (STRs) are the most utilized
fermenters at both lab and industrial scale.
 The two most commonly used fermenter
configurations for microbial cultivation are the
continuous STR (CSTR) and the air-lift reactor
(ALR).
 Other fermenter configurations have been used
for EPS production by different microorganisms.
For example, continuous production of levan in a
packed-bed bioreactor (Freitas et al., 2017).

19. Downstream processing/ Recovery
 The specific method used for recovery of EPS from
the cultivation broth depends on characteristics of
the producing organisms, the type of
polysaccharide and the desired degree of purity.
 The downstream processing involves several steps,
 Starting with cell removal by centrifugation or filtration,
 Recovery of the polymer from the cell- free
supernatant.
 Precipitation of the polymer by addition of a water-miscible
non-polar solvent, such as acetone, ethanol or isopropanol.
 The precipitate can then easily be separated from the solvent-
water mixture and dried.

20. Downstream processing/ Recovery
 Several additional procedures can be used to
remove contaminants, namely re-precipitation
with diluted aqueous solutions, deproteinization
by chemical or enzymatic methods and membrane
processes (Kumar, Anandapandian, & Parthiban,
2011; Finore et al., 2014; Freitas et al., 2017).

21. Examples: EPS produced by the native Leuconostoc
pseudomesenteroides (Paulo et al., 2012).
 For the extraction of exopolysaccharides, after the incubation
period, the culture is homogenized
 centrifuged
 The pelleted material is discarded, and absolute alcohol (1:2)
added
 stored in the refrigerator
 The EPS precipitates are separated using decantation flasks.
 Each precipitate is partially purified by conducting three
successive washes in distilled water, followed by reprecipitation
in absolute alcohol
 Subsequently, the precipitates can undergo dialysis in distilled
water by adding in membranes with an exclusion limit of 15 kDa
 The EPS precipitates were dried in an oven to a constant weight
 The EPSs in the form of powder are stored in airtight glass jars

22. Example: Production, extraction and
purification of microalgae EPS
(Delattre et al., 2016).

23. Example: Production of xanthan gum

24. Example: Processing of
pullulan

25. Production Yields
 Depending on the species and the cultivation
conditions, EPS production by bacteria may range
between 0.29 and 100 g/L, in processes taking 0.5
– 7 days (Freitas et al., 2017).
 Fungi usually have longer cultivation times (2 - 32
days) than bacteria (0.5 – 7 days), which in some
cases translates into lower volumetric
productivities (Freitas et al., 2017)

26. Scale up
Elective methods for improving the
commercial scale production and field
application of microbial biopolymers are;
 optimizing the fermentation conditions,
 biotechnological tools involving genetic and
metabolic engineering,
 the exploration of cheap fermentation
substrates for their production (Sanlibaba &
Çakmak, 2016).

27. Application
 According to Shukla, (2017) and Sanlibaba & Çakmak,
(2016) Bacterial EPSs have possible commercial
applications in
 pharmaceutical industry,
 food processing,
 drug detoxification,
 bioremediation,
 cosmetics
 Bioflocculants,
 bio-absorbents,
 heavy metal removal agents,
 drug delivery agents,

28. Uses in the food industry
 In the food industry microbial EPSs can be used in
 control viscosity and modify flow
 Improve texture, mouth feel and freeze-thaw stability,
 Thickeners,
 Suspending agents,
 Low calories food products,
 Dietary fibers
 Films and coating agents,
 Salad dressings,
 Frozen food icing,
 Moisturizing agents

29. Conclusion
 EPSs are secreted by microorganisms for their
survival in harsh environmental conditions
especially for protection.
 Different microbial species can produce EPS
depending on the cultivation conditions
 Bacterial EPSs have possible commercial
applications in in may industrial processes

30. Thanks