Maria A. M. Reis
REQUIMTE/CQFB, Department of Chemistry,
FCT/Universidade Nova de Lisboa, Portugal
Ciência 2009, 30 de Julho de 2009
73100
Plants
Guar Gum
Microorganisms
Arabic Gum
Xanthan
Starch
Gellan
Pectins
Pullulan
Bacterial Alginate
Algae
Carrageenan
Alginate
higher growth rates
more amenable to
manipulation of conditions
Animals
Chitin/Chitosan
new / improved properties
high substrate costs
Low-cost Substrates:
Agricultural and Industrial
wastes and byproducts
Glycerol
Biodiesel production in Europe (thousand tons)
6000
5000
4000
3000
2007:
2000
• Europe ~600.000 tons glycerol
1000
• USA:
0
1998
2000
2002
2003
Source: European Biodiesel Board
Ciência 2009, 30 de Julho de 2009
2004
2005
2006
2007
>120.000 tons glycerol
Production of extracellular bacterial polysaccharides (EPS)
from glycerol byproduct from the biodiesel industry
Extraction and purification of the EPS
Characterization of the EPS
Applications of the EPS
Reis et al. (2008) Patent WO 2008/127134
Culture medium
K+, Mg2+, Fe2+, Mn2+, Ca2+,
Mineral salts
Co2+, Cu2+, Zn2+, SO42-, ClNitrogen source
(NH4)2HPO4
Carbon source
pure glycerol
glycerol byproduct
Operation conditions
Temperature
30ºC
pH
6.8±0.5
DO
<20%
Aeration
<0.5 vvm
Stirring speed
400 – 800 rpm
Feeding solution:
high C/N ratio
residual N concentration
Ciência 2009, 30 de Julho de 2009
Pseudomonas oleovorans
40
Feeding solution
12
30
8
20
4
10
0
0
0
1
2
3
Time (day)
Ciência 2009, 30 de Julho de 2009
4
5
6
Glycerol (g l-1)
Ammonium (g l-1 )
CDW (g l-1 )
EPS (g l-1 )
16
Culture broth viscosity
viscosity built up concomitant
105
with EPS production
day 5
104
day 4
103
h (Pa.s)
culture broth develops
non-Newtonian characteristics,
day 3
102
acting as a pseudoplastic fluid
day 2
101
100
10-1
day 1
loss of homogeneity in the bioreactor
100
101
g
Ciência 2009, 30 de Julho de 2009
(s-1)
102
(mixing, mass, oxygen and heat transfer)
Polymer
EPS
EPS
Productivity
Yield
(g L-1)
(g L-1 d-1)
(g g-1)
Glycerol /
7 – 18
2.4 – 4.8
0.15 – 0.40
Glycerol byproduct
15 – 30 1
16 – 30 1
---
---
US4692408
2.7 – 15.3 (a)
---
0.8 – 0.9
GB2008138
10.5 – 30.0
3.1 – 12.2
0.34 – 0.81
García-Ochoa et al, 2000
0.6 – 3.5
0.15 – 1.75
---
US3856625
1.3 – 4.6
0.43 – 1.53
0.25
Peña et al, 2000
Microorganism
Substrate
Pseudomonas
oleovorans
Xanthan
Xanthomonas
gum
campestris
Glucose / Sucrose
Bacterial
Azotobacter
alginate
vinelandii
Sucrose
1
Ciência 2009, 30 de Julho de 2009
crude polymer; (a) Continuous process
References
Present study
biomass
H2O
cell-free
supernatant
ethanol
Native EPS
protein, nucleic acids,
salts, and other
molecules removal
treated
supernatant
ethanol
ethanol
Semi-purified EPS
Purified EPS
Downstream costs
• pet food
• animal feed
• wastewater treatment
• mining industry
Ciência 2009, 30 de Julho de 2009
• cosmetics
• food
• paints
• paper
• petroleum industry
• cosmetics
• pharmaceuticals
• food
• paints
• paper
Chemical composition
 Carbohydrates
Galactose
50 – 90 %mol
Glucose
1 – 25 %mol
Mannose
1 – 25 %mol
Rhamnose
0.5 – 20 %mol
Other components
1.7 – 11.0 %wt
 Ash
(K+, Na+, Mg2+, Fe2+,Ca2+)
1 – 15 %wt
 Protein
 Acyl groups
Acetyl
0 – 6.18 %wt
Pyruvil
0.02 – 4.50 %wt
Succinyl
0 – 2.30 %wt
Solubility
 insoluble in organic solvents
(hexane, ethyl acetate, toluene,
chloroform, etc.)
Molecular weight
1.0 – 5.0 x 106
 highly viscous aqueous dispersions
Freitas et al. (2009) Bioresource Technology, 100, 859-865
Ciência 2009, 30 de Julho de 2009
Rheology
 Pseudoplastic fluid behaviour
 Newtonian behaviour at low shear rates
Polymer concentration: 10 g L-1 in 0.1M NaCl, 25ºC
10 3
Xanthan
10 2
EPS
Guar gum
h (Pa.s)
10 1
CMC
similar to Guar gum
Alginate
10 0
better viscosity enhancing properties
than CMC and alginate
10 -1
10 -2 -2
10
10 -1
10 0
10 1
g (s -1 )
10 2
10 3
10 4
Hilliou et al. (2009) Carbohydrate Polymers (in press)
(doi: 10.1016/j.carbpol.2009.05.011)
Ciência 2009, 30 de Julho de 2009
less viscous than xanthan
at low shear rates
Rheology
0.25
Viscosity at shear rate of 5 s-1 (Pa.s)
Viscosity at shear rate of 5 s-1 (Pa.s)
101
100
10-1
10-2
0.0
Cooling
0.15
0.10
0.05
0.00
0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
Heating
0.20
20
1.6
EPS concentration (wt%)
Freitas et al. (2009) Carbohydrate Polymers (in press)
(dx.doi.org/10.1016/j.carbpol.2009.05.016)
Ciência 2009, 30 de Julho de 2009
40
60
Temperature (ºC)
(0.8% EPS solution)
80
100
Rheology
Viscosity at shear rate of 5 s-1 (Pa.s)
1.2
1.0
0.8
0.6
0.4
0.2
0.0
15
25
35
45
65
Temperature (ºC)
80
2.9
5.3
7.1
10.1
pH
(1.2% EPS solution)
Ciência 2009, 30 de Julho de 2009
11.8
0.0
0.1
1.0
NaCl (M)
2.0
Emulsion forming and stabilizing capacity
E24 for 0.8% EPS solutions against
several hydrophobic compounds
6 mL hexadecane +
4 mL EPS dispersion (wt%)
Emulsifying index (%)
0
9
22
26 28 60
87
100
Hydrocarbons
E24 (%)
n-hexadecane
70
n-hexane
cyclohexane
0
0.05 0.1 0.2
0.4 0.6 0.8 1.0
EPS concentration (%)
n-decane
xylene
diethylether
similar to xanthan gum
much higher emulsifying activity
than Guar gum, CMC or alginate
chloroform
benzene
toluene
Ciência 2009, 30 de Julho de 2009
Oils
E24 (%)
Paraffin oil
30
Silicone oil
<10
Cedarwood oil
30
Palm oil
<10
Sunflower oil
20
Corn oil
<10
Olive oil
65
Oleic acid
<10
75
40
35
60
25
70
60
65
Emulsion forming and stabilizing capacity
80
Emulsification index (%)
70
60
50
40
30
20
10
0
20
30
40
50
Temperature (ºC)
60
0
2
4
6
8
10
pH
(0.6% EPS solution
+ hexadecane)
Ciência 2009, 30 de Julho de 2009
12
0.0
0.4
0.8
1.2
NaCl (M)
1.6
2.0
Flocculating capacity
(polymer dispersions: 0.1 wt%)
100
Flocculating activity (%)
80
60
40
20
0
CMC
EPS
Ciência 2009, 30 de Julho de 2009
Guar
gum
Xanthan
Alginate
40 mL kaolin suspension
+ 5 mL CaCl2 (10 wt%)
+ 0.25 mL polymer dispersion
+ H2O (up to 50 mL)
Film-forming capacity
 flexible and transparent
 hygroscopic
 permeable to water vapour
WVP similar to corn starch and pectin films
 rather stiff
mechanical properties similar to corn starch films
stress at break = 30 MPa
elongation = 3%
Young modulus = 1200 MPa
Film properties improvement:
plasticizers
blends with other polymers
Ciência 2009, 30 de Julho de 2009
Emulsifying activity
foods
cosmetics
paints
bioemulsifier
biosurfactant
Reology
thickening, binding,
texturing and stabilizing
agent in liquid systems
foods
pharmaceuticals,
cosmetics
paints, paper, and
petroleum industries
Flocculating activity
bioflocculant
agent
water and wastewater
treatment, foods,
mining industry
Film-forming capacity
biodegradable
films
barriers
encapsulation
packaging
Ciência 2009, 30 de Julho de 2009
Microbial extracellular polysaccharide produced from glycerol
low cost substrate
valorization of glycerol byproduct
new biopolymer
Simple EPS extraction procedure
EPS physical-chemical characteristics:
 high molecular weight
Emulsifying activity
 anionic heteropolysaccharide
Flocculating activity
 amorphous
 insoluble in organic solvents
 viscous aqueous dispersions
Ciência 2009, 30 de Julho de 2009
Film-forming agent
73100
Team:
Rui Oliveira,
Filomena Freitas
Vítor D. Alves,
Cristiana Torres,
Mónica Carvalheira
Nuno Costa
Ciência 2009, 30 de Julho de 2009