MICROBIAL BIOPOLYMER

1. Pullulan
2. Gellan Gum
3. Dextran
4. Xanthan Gum

Pullulan
Definition
Structure
Microorganism
Properties and quality parameters
Production and Downstream Process of
Pulullan
Pullulan derivatives
Application
Pullulan
Pullulan is a polysaccharide polymer
consisting of maltotriose units,
also known as -1,4- ;-1,6-glucan
Three glucose units in maltotriose are
connected by an -1,4 glycosidic bond,
whereas consecutive maltotriose units
are connected to each other by an -1,6
glycosidic bond.

Pullulan Structure

Optimum temperature : 26-28oC, pH 5 -6.5
Aureobasidium pullulan
Aureobasidium pullulan
Source of A. pullullans isolates
Wood
leather
hydrocarbons
synthetic materials
paper (paper, paper pulp)
human skin
paint (watercolours)
plants
food products (wheat, oats and bean seeds,
flours, fruits and vegetables, fruit juices)
soil
textile (cotton)

Aureobasidium pullulans
in leather (flesh)
Aureobasidium pullulans
in leather (grain)
Aureobasidium pullulans
(cotton)
Aureobasidium pullulans
in acidic paper
Growth curve of A. pullulan
On Malt-Agar growth medium
(MA) (pH 6.5)
On CYA growth medium (pH 5.5) Slimy
colonies (final pH 6).
On Czapek growth medium (initial pH
5.5 and final 5.5)
Properties and quality
parameters
1. Water soluble, insoluble in organic solvents, non
hygroscopic, aqueous solutions are stable and show a
relatively low viscosity compared to other
polysaccharides.
2. It decomposes at 250-280C. It forms easily, thermo-
stable, transparent, elastic, antistatic films, with an
extremely low oxygen permeability (compared to celofan,
polypropylene, polyesther, polyvinylchloride, etc)
3. Moldable, and spinnable, good adhesive and binder.
4. Non-toxic, edible, biodegradable and biocompatible.
Production and Downstream
Process of Pulullan in general
Submerged fermentation using carbohydrate as main media,
nitrogen source, minerals, temperature of 28o C, agitation
of 200 rpm

Downstream processing of fermentation broth includes :
biomass separation precipitation of filtrates by solvent
2- propanol concentration purification of solution
containing the pullulan-type polysaccharide ( partially
purified product or a highly-purified one modern
ultrafiltration techniques are used).
FERMENTATION STEPS
A.pululan in complex nitrogen
agar plates with 50 g/L sucrose
1
st
Media
propagation : malt
extract broth
2
nd
Media
propagation :
complex nitrogen
broth containing 50
g/L sucrose.
Fermentation
Centrifugation
and separation
of biomass
Precipitation of
pullulan by 2 propanol
followed by further
purification
Drying
Milling Packing
Examples of Pullulan Medium
Composition
Complex Nitrogen medium
5 g KH2PO4, 1 g NaCl, 0.6 g (NH4)2SO4, 0.2
g MgSO47H2O, 1 g yeast extract, 0.01 g
FeSO4, 0.01 g MnSO4 and 0.01 g ZnSO4 in
grams/liter of water
malt extract broth :20 g malt extract, 20 g
glucose and 1 g peptone per liter of water
(ATTC Medium 325).
Factors affecting pullulan
production
Media components
Strain of microorganisms
Characteristic of microorganism : the phylamentous
forms or chlamydospores are less productive than
the yeast or pigment-free blastospores (Audet et al.,
1996).
According to Shabtai and Mukmenev (1995), the
yeast-like cells did not produce much pullulan in the
presence of pigment, and the non pigmented swollen
blastospores or germinating blastospores help to
trigger the elaboration of pullulan
Commercial pullulan products
pullulan derivatives
Crosslinked pullulan microparticles
Carboxymethyl pullulan
Sulfopropyl pullulan
Pullulan acetate
Crosslinked pullulan
microparticles

Crosslinking reaction between pullulan and
epichlorohydrin followed by separation,
washing, drying
Healing of infected wounds

a b c
Treatment of a leg wound (horse) with crosslinked
pullulan microparticles: a) infected wound; b) wound
after several applications of crosslinked pullulan
microparticles; c) wound after 2 weeks of treatment.
Crosslinked pullulan microparticles in dry state
Application
edible films for food packaging
breath freshener or oral hygiene products
such as Listerine Cool Mint PocketPaks.
As a food additive, it is known by the E
number E1204 (e.g in cosmetics (hydrating
creams and gels)
filming agent in pharmaceutics (retard
tablets, capsules and microcapsules)
Entrap colours and flavours
Innovative use of pullulan film in
sugar confectionary
Effect of pullulan coating in tablet
Soft candy manufacturing
Pullulan capsules
Application (cont.)
Textiles industry (antistatic, in non-
woven)
Paper industry (coating, in composition)
Photosensible materials (emulsions)
Agriculture (low release fertilizers)
Metalurgy (foundery mold or casting)
Mining (floculant, binder).

Definition
Structure
Microorganism
Production and downstream process
Application
Gellan Gum
a water-soluble polysaccharide produced
by a bacterium Pseudomonas elodea
used primarily as a gelling agent,
alternative to agar in microbiological
culture.
MW : 500 000
Forming viscous solution, insoluble in
ethanol
Structure
The repeating unit of the polymer is a
tetrasaccharide which consists of two residues
of D-glucose and one of each residues of L-
rhamnose and D-glucuronic acid.
The tetrasacharide repeat has the following
structure:
[D-Glc(14)D-GlcA(14)D-Glc(14)L-
Rha(13)]n.
As it is evident from the formula the
tetrasacharide units are connected with each
other using an (13) glycosidic bond.
Molecular structure
Structure (cont.)

Microorganism
Pseudomonas elodea = Sphingomonas
elodea
The composition and structure of native
gellan gum produced by commercial
fermentation is identical to the naturally
occurring polysaccharide formed by
Sphingomonas elodea on plants of Lily
pad varieties.
Pseudomonas elodea
Sphingomonas
Gram-negative, rod shaped, chemo
heterotrophic,
strictly aerobic bacteria
containing glycosphingolipids (GSLs) in
their cell envelopes,
typically produce yellow-pigmented
colonies
Yellow pigmented colonies of
Sphingomonas
Properties of gellan gum
ability to suspend while contributing
minimal viscosity via the formation of a
uniquely functioning fluid gel solution
with a weak gel structure.
setting temperature, degree of structure
and thermal stability.
Water soluble
Biosynthesis
1. intracellular formation of the nucleotide-
sugar precursors, UDP-Glc, UDP-GlcA, and
dTDP-l-Rha.
2. formation of the repeat unit, with sequential
transfer of the sugar donors to an activated
lipid carrier by committed
glycosyltransferases
3. gellan polymerization
4. Export to outer of cells

Production and downstream
process in general
produced by a pure culture fermentation of
carbohydrates by Pseudomonas elodea,
purified by recovery with alcohol, dried, and
milled.
The direct fermentation of sweet cheese whey
diluted 1:5 with water resulted in production of
approximately 7 g of EPS (extra
polysaccharides Sellular ) gellan gum per liter
and in a 70% reduction in the initial BOD5
interesting valorisation of this waste of the
dairy industry and BOD reduction.
Factors affecting gellan gum
production
1. Media components
Molasses 112.5 g/L, tryptone 1 g/L,
casamino acid 1 g/L , disodium hydrogen
orthophosphate 1 g/L and manganese
chloride 0.947 g/L produced gellan
gum of 13.81 g/L

Factors affecting gellan gum
production (cont.)
2. Addition of precursor The sugar
nucleotides providing the activated
precursors for synthesis of
tetrasaccharide i.e UDP-glucose, TDP-
rhamnose and UDP-glucuronic acid.
3. Addition of amino acid triptophan
enhanced cell growth
4. pH 6.5 - 7



Factors affecting gellan gum
production (cont.)
5.Agitation rate : 250 rpm using a helical
ribbon impeller
6. Higher DOT (dissolved oxygen tension)
levels improve the viscosity and
molecular mass of the polymer
7. Temperature : 30oC
DOWNSTREAM PROCESS

Fermentation
broth
Heating 90-95oC, 10-15
min
Killed bacteria and reduced the viscosity
Dilution
Centrifugation and filtration
Separates cells and filtrates
Precipitation by alcohol
followed by centrifugation
Drying 55oC
Milling and packing of
crude gellan gum
Purification of gellan gum
After alcohol precipitation, the products are
washed repeatedly with acetone and ether,
dissolved in deionised water and dialyzed
against deionised water by using dialysis
tubing with molecular mass cut-off of 12 000
14 000.
After dialysis for 23 days with four or five
changes of deionised water, the solution was
lyophilized to formulate dry gellan powder
gel filtration chromatography (GFC) can also
be used
Application
Applications in foods : stabilizer,
emulsifier, thickener
Applications in pharmaceutical industry
Solid culture media for growth of
microorganisms and plants
Gel electrophoresis in biological
research
Cell immobilization
DEXTRAN
complex, branched glucan
(polysaccharide made of many glucose
molecules) composed of chains of
varying lengths
Dextran was first discovered by Louis
Pasteur as a microbial by product in
wine
Structure
The straight chain consists of -1,6
glycosidic linkages between glucose
molecules, while branches begin from -
1,4 linkages (and in some cases, -1,2
and -1,3 linkages as well).
molecular weights ranging from 10,000
Da to 150,000 Da.
Molecular Structure
Microorganisms
Dextran is synthesized from sucrose by
certain lactic-acid bacteria, the best-known
being Leuconostoc mesenteroides and
Streptococcus mutans, L. dextranicum , S.
salivarius, S. bovis
Dental plaque contains dextran

Leuconostoc mesenteroides Streptococcus mutans

Slimy dextran produced by Leuconostoc
mesenteroides CMG713 on sucrose
containing medium with 0.005 % sodium
azide
Production of dextran

Medium : using sucrose as carbon source, examples :
Sucrose 150.0 g., Peptone 2.5 g., Yeast extract 2.5 g.,
K2HPO4 , 5.0 g., NaCl 2.5 g., and a water extract of
sugar refining charcoal 2.0 ml.
Sucrose 150.0 g., acid hydrolyzed casein 5.0 g., Yeast
extract 1.0 g., K2HPO4 5.0 g., NaCl 2.0 g., and MgSO4
0.022 g.
(g l1): sucrose, 150.0; bacto-peptone, 5.0; yeast extract,
5.0; K2HPO4, 15.0; MnCl2.H2O, 0.01; NaCl, 0.01; CaCl2,
0.05.
Condition and downstream
process
Condition : 30 oC, 20 hours

Downstream process :
The culture medium after 20 hours was precipitated
using equal volume of chilled ethanol, shaken
vigorously, centrifuged at 10,000 rpm for 15 minutes
and the supernatant was decanted. This step was
repeated twice.
The precipitated dextran was dried under vacuum
over calcium chloride at 30C. The dextran yield was
calculated on dry weight basis.
Purification
For removal of impurities, dextran obtained
from precipitation was dissolved in distilled
water.
The dextran slurry was again precipitated with
equal volume of chilled ethanol. This
procedure of re-dissolving, precipitation and
washing was repeated three times to remove
cells debris.
Purified dextran was dried under vacuum over
calcium chloride at 30C
Factors affecting dextran
production
1. Incubation time
Factors affecting dextran
production (cont.)
2. Substrate concentration

Factors affecting dextran
production (cont.)
3. Temperature

Factors affecting dextran
production (cont.)
4. pH
Molecular weight of dextran types
using glass column chromatography
packed with Sepharose CL6B.
Remarks :
Blue Dextran, 2000,000;
Industrial dextran, 5000,000-40,000,000;
Dextran from Leuconostoc mesenteroides
CMG713, 5,000,000-20,000,000.
Application medical uses
eye drops as a lubricant
decrease vascular thrombosis.
Dextran in intravenous solution provides an
osmotically neutral fluid that once in the body is
digested by cells into glucose and free water.
It also increases blood sugar levels.

Other uses
size-exclusion chromatography matrices;
an example is Sephadex.
bead form to aid in bioreactor
applications (immobilization)
stabilizing coating to protect metal
nanoparticles from oxidation
make microcarriers for industrial cell
culture
Application (cont. )
GUM XANTHAN
ISI KULIAH :
1. Deskripsi Produk ( Sifat fisiko kimia, biosintesa ,
kegunaan dalam industri, dll)
2. Mikrooorganisme
3. Tahapan fermentasi
4. Proses Hilir (recovery)
5. Prospek Pengembangan
di Indonesia
Gum xanthan : eksopolisakarida mikrobial yang diproduksi
oleh Xanthomonas campestris dari bahan
berkarbohidrat
Sifat Fisiko Kimia :
1. Heteropolisakarida anionik yang bercabang
2. Mengandung D-glukosa (2.8 mol), D-manosa (3.0 mol),
D-glukuronat (2.0 mol), asam asetat dan asam piruvat
3. Rantai utama mirip kerangka selulosa (rantai glukosa
berikatan 1,4)
4. Rantai sisi mengandung dua unit manosa dan satu unit
asam glukuronat
5. Asetat dan piruvat berikatan pada ujung manosa
6. BM : 2-50 x 10
6
atau 3 x 10
7
dalton


Struktur molekul gum xanthan
TURUNAN KIMIA DAN KOPOLIMER CANGKOKAN GUM
XANTHAN
1. Gum Karboksimetil xanthan
2. Gum dietilaminotil xanthan
3. Ester propilenglikol xanthan
4. Gum xanthan sulfat
5. Ikatan silang aldehid gum xanthan
6. Gum deasetil xanthan
7. Gum xanthan - g poli akrilamida

Sampai saat ini tidak ada yang diproduksi secara komersil

SIFAT-SIFAT GUM XANTHAN :
1. Viskositas tinggi pada konsentrasi rendah,
2. Kekenyalan semu (pseudoplasticity) tinggi
3. Mudah larut dalam air panas maupun dingin
4. Viskositas larutan gum xanthan stabil terhadap suhu
5. Viskositas larutan gum xanthan stabil terhadap pH
6. Kelarutan dan kestabilan tinggi pada asam
7. Unggul dalam daya suspensi karena yield valuenya tinggi
8. Kesesuaian yang baik dengan berbagai garam
9. Stabilitas pada proses pelelehan dari keadaan bekunya
(freeze thaw)


SIFAT GUM XANTHAN (LANJUTAN)
10. Kompatibel terhadap beberapa pelarut seperti methanol,
ethanol, isopropanol dan aseton sampai konsentrasi 50-60
%. Konsentrasi pelarut > 60 % akan menyebabkan
gelatinisasi atau pengendapan gum xanthan
11. Tidak terdegradasi oleh enzim protease, selulase
hemiselulase, pektinase dan amilase, tetapi dapat
terdegradasi oleh pengoksidasi kuat seperti peroksida,
persulfat dan hipoklorit
12. Sinergi terhadap gum yang lain seperti gum guar, dan
gum dari biji locust (locust bean gum) serta galaktomanan


Viskositas (cp)
1000
100
0.5 1.0 1.5 2.0 2.5 3.0 Konsentrasi (%)
Pengaruh konsentrasi gum xanthan thd viskositas
Konsentrasi garam (NaCl) (%)
Viskositas (cP)
100
1000
0.01 0.05 0.1 1.0
0.1 % gum xanthan
0.25 % gum xanthan
0.5 % gum xanthan
1.0 % gum xanthan
Pengaruh konsentrasi garam thd viskositas lar gum xanthan
Temperatur (oF)
Viskositas (cP)
1000
10000
50 100 150 200
0.1 % gum xanthan
0.25 % gum xanthan
0.5 % gum xanthan
1.0 % gum xanthan
Pengaruh suhu thd viskositas lar gum xanthan
pH
Viskositas (cP)
1000
10000
1 3 5 7 9 11 13
0.1 % gum xanthan
0.25 % gum xanthan
0.5 % gum xanthan
1.0 % gum xanthan
Pengaruh pH thd viskositas lar gum xanthan
Viskositas (cP)
Laju geser detik
-1
0.1 1 10 100 1000 10 000
1
10
100
1000
10 000
100 000
1 000 000
0.5 %
1 %
2.5 %
Pengaruh laju geser terhadap viskositas larutan gum xanthan
Guar gum
Tara gum
Sifat sinergi gum xanthan dengan
polisakarida lainnya

APLIKASI DALAM INDUSTRI :
1. Industri Petroleum :
a. Sebagai cairan pelicin dalam pengeboran sumur minyak
b. Sebagai cairan untuk mengikat dan memisahkan garam-garam
dari hasil pengeboran minyak lepas pantai dari cairan petroleum
yang diinginkan
c. Sebagai cairan pemecah (fracturing fluid ) berbasis air
d. Untuk mempercepat pengambilan minyak (oil recovery)

Oil drilling
2. Industri Kimia dan lainnya
a. Pakan ternak : sebagai cairan pelengkap pakan (Liquid feed
supplements)
b. Flowable pesticides
c. Pewarnaan dan pengecatan tekstil
d. Pelapis keramik
e. Pembersih
f. Pensuspensi cairan tinta, cat dan perekat kertas

3. Industri Pharmasi dan Kosmetika :
a. Stabilizer emulsi cream untuk obat dan kosmetika
b. Pelarut cream dan lotion
c. Pensuspensi pasta gigi atau larutan pencuci gigi
4. Industri pangan :
a. Pastry filling
b. Penstabil saus dan kaldu
c. Pengemulsi dressing
d. Pengemulsi dan stabilizer dairy products
BIOSINTESA
Ada 4 jenis enzim yang terlibat :
1. Enzim untuk metabolisme awal substrat : hexokinase
2. Enzim untuk sintesis dan interkonversi nukleotida gula (UDP
glucose phosphorylase)
3. Enzim untuk pembentukan pengulangan unit polimer
(monosakarida) (transferase)
4. Enzim polimerase untuk pembentukan biopolimer
eksopolisakarida
Tahapan biosintesa :
1. Metabolisme substrat karbohidrat
2. Sintesis dan interkonversi nukleotida gula
3. Pengulangan unit monomer
4. Polimerisasi
MIKROORGANISME :
Bakteri penghasil gum xanthan :
Xanthomonas campestris
1. Gram Negatif
2. Membentuk koloni berlendir
berwarna kuning
3. Diisolasi dari daun/tanaman
yang berpenyakit,misalnya daun
kedelai yang terkena pustule,
daun kubis, tebu, dll
TAHAPAN FERMENTASI
Xanthomonas campestris
Pengembangan
inokulum
Tangki benih
Fermentasi
Pasteurisasi
dan
pemisahan
biomassa
Pengambilan
gum dengan
pelarut
Pengeringan
Penggilingan Pengemasan
Komponen media :
- Karbohidrat sbg sumber C
- Sumber N
- Mineral
- Antifoam
KONTROL PROSES
Level of control

Metabolic





Environmental Nutrients, pH, P O2, P CO2, shear, temperature, fluid properties


Equipment design
operation Nutrients, acid/base, antifoam addition, air flow, speed, over
pressure, coolant, flow rate and temperature, sparger, and fermenter
impeller geometry
Product biosynthesis
Cell environ
Other processes
Product
Waste (cells, other
products, heat)
SIFAT RHEOLOGI CAIRAN FERMENTASI GUM
XANTHAN SANGAT KHAS :
1. Pada penggunaan bioreaktor berpengaduk akan
timbul zona stagnasi
2. Masalah ini diatasi dengan penggunaan airlift
bioreactor

Air lift bioreactor
Stirred tank bioreactor
PROSES HILIR :

Cairan fermentasi
Pasteurisasi Untuk membunuh sel bakteri yang masih tersisa
Pengenceran
Untuk memudahkan pemisahan biomassa
karena kekentalannya telah diturunkan
Sentrifugasi
Untuk memisahkan sel/biomassa
Pengambilan gum dg pelarut Untuk mempresipitasikan gum , bisa dg
etanol, isopropanol, metanol, dll
Pengeringan
Penggilingan
BEBERAPA MERK GUM XANTHAN KOMERSIL :
1. Keltrol
2. Kelzan
3. Jungbunzlauer
4. Degussa
5. dll
Prospek Pengembangan di Indonesia :
1. Ketersediaan bahan baku : berbagai jenis sumber
karbon dan nitrogen
2. Ketersediaan sumber isolat
3. Aspek teknis teknologis (penggunaan teknologi
fermentasi)
4. Aspek pasar yang terbuka luas