5th World Congress on Biotechnology, Valencia, Spain
June 25th -27th , 2014
Production and Application of Lipopeptide Biosurfactant for
Dispersing Oil Spill in Seawater
Suwat Soonglerdsongpha1, Witchaya Rongsayamanont2, Nichakorn Khondee2,
Onruthai Pinyakong2, Ekawan Luepromchai2
1
Environmental Research and Management Department, PTT Research and Technology Institute, Thailand
2 Department of Microbiology, Faculty of Science, Chulalongkorn University, Thailand
1
Biosurfactant
Biosurfactants are surface-active biomolecules
produced by microorganisms.
Hydrophilic Moiety
• Acid
• Peptide cations or anions
• Mono-, di- or polysaccharides
Biosurfactant
Hydrophobic Moiety
• Unsaturated or saturated
hydrocarbon chains
• Fatty acids
Hydrophilic Moiety
Hydrophobic Moiety
(Surfactant head)
(Surfactant tail)
CMC
(http://www.ualberta.ca/~csps/JPPS8(2)/C.Rangel-Yagui/solubilization.htm)
CMC = Critical Micelle Concentration
(http://people.umass.edu/mcclemen/Group.html)
2
Biosurfactant
Classification of Biosurfactants
Glycolipids – Sophorolipids, Trehalolipids, and Rhamnolipids
Lipopeptides and Lipoproteins
Fatty acids
Phospholipids
Neutral lipids
Polymeric biosurfactants
Particulate biosurfactants
Rhamnolipid
Sophorolipid
Trehalolipid
Surfactin (Lipopeptide)
http://biotechsupportbase.com/wp-content/uploads/2014/02/surfactants.png
3
Biosurfactant
Advantages
 Non-toxic or low in toxicity
 Biodegradable
 Wastes can be used as raw materials
 Able to work at critical condition
 Wide applications
Limitations
 Low production yield
 High production cost
4
Applications of Biosurfactants
Biosurfactant applications
1.
2.
3.
4.
5.
6.
7.
8.
Household detergent
Personal care
Industrial cleaner
Food processing
Oilfield chemicals
Agriculture chemicals
Textiles
Others
Environmental applications
 Soil bioremediation
 Oil dispersant
Biosurfactant market volume share (by application, 2013)
(http://www.grandviewresearch.com/industry-analysis/biosurfactants-industry)
5
Dispersant use for oil spill
 Blend of two or three surfactants
 Ex. nonionic /anionic/solvent
Oil droplets
Increase natural attenuation and
biodegradation by microorganism.
However, synthetic dispersants are
usually toxic and may decrease
biological activity of microorganisms.
6
Objectives
Low cost substrates???
Substrates
Microorganisms
Biosurfactant producing
Microorganisms???
Production & downstream
processes
Production & downstream
Processes???
Biosurfactants
Dispersing oil
application???
7
Scope of Experiment
Isolation of biosurfactantproducing bacteria
Evaluation of substrate
utilization
Production of biosurfactant by chitosan-immobilized cell
Purification of biosurfactant by Foam fractionation and Freeze-drying
Biosurfactant
Characterization of
biosurfactant
Properties & stabilities of
biosurfactant
Application of biosurfactant
for dispersing oil
8
Biosurfactant producing microorganisms &
Substrate selection
Isolation of microorganisms
ดิน
ทราย
นา้ ทะเล
นา้ คลอง
ดินป่าชายเลน
ี
นา้ เสย
ใบไม้
คล ังเก็บจุลน
ิ ทรีย ์
107
species
Substrate Selection
Pure substrate
 Glycerol
 Soybean oil
 Palm oil
 Crude oil
 Diesel
 Lube
Microbial
screening
Organic waste
Surface tension
< 40 mN/m
58 species
 Slop oil
 Bottom glycerol
 High yield
 Low cost
 Waste reduction
-PTT Group Use only-
Bacillus sp.
Highest yield
GY19
9
Screening of biosurfactant-producing bacteria
107
strains
Surface tension
< 40 mN/m
Highest yield
Pure glycerol
Yield
Bacillus sp.
0.13 g/l
GY19
58 strains
20 genus
Bottom glycerol
+ palm oil
Leu CH3
Surfactin
O Glu
myristoleic acid
O
HO
NH
O
O
NH
H3C
Leu
CH3 O
H3C
N
H
CH3
O
HN
O
O
H
N
H
N
O
CH3
O
CO2H
Surfactin
Leu
CH3
Yield 2.8 g/l
CH
NH 3
CH3
CH3 Val
Lipopeptide biosurfactant
Asp
Leu
Biosurfactant from pure
glycerol
BSF PG
10
Lipopeptide production and recovery processes
Substrate
Low cost
substrate
Waste
utilization
Waste from biodiesel
production
Production
Downstream
High production yield
Efficient recovery and
purification methods
Immobilized bacteria on
cheap and effective
materials
Foam fractionation
 Solvent-free method
Develop
Cheap, easy and
effective bioprocess
Bottom glycerol
(10% glycerol + fatty
acids and etc.)
Chitosan-immobilized cells
Foamate  easy to use
and effective for further
application
Foam fractionation
11
Lipopeptide production by chitosan immobilized cells
in stirred tank bioreactor
- Palm oil was added as precursor
- Maximum lipopeptide yield 6.65 g/L
Crude biosurfactant
10
9
8
7
6
5
4
3
2
1
0
Oil
Glycerol
80
Cycle
1
Cycle
3
Cycle
2
Cycle
4
Cycle
5
Cycle
6
60
40
20
Remaining glycerol (g/L)
Crude biosurfactant (g/L)
Remainging oil (g/L)
A
0
0
5
10
Time (day)
15
20
25
12
Properties of lipopeptide biosurfactant
Freeze-drying
Cell free supernatant
Foam-fractionation
Foamate
Biosurfactant powder
50% lipopeptide (w/w)
70
Lowest SFT ~ 26 mN/m
Concentration of foamate (%)
Surface tension (mN/m)
60
1st batch production
2nd batch production
50
3rd batch production
40
30
20
10
0
0.001
0.01
0.1
1
Biosurfactant powder conc. (%, w/v))
10
13
40
surface tension (mN/m)
surface tension (mN/m)
Stabilities of lipopeptide biosurfactant
35
30
25
20
15
10
surface tension (mN/m)
30
40
50
60
Temperature (C)
70
80
40
35
30
25
20
15
10
0
1
2
3
4 5 6
NaCl (%)
7
8
9 10
40
35
Surface activity was stable at
30
25
 Temperature (40-100oC)
20
 Electrolytes (> 6% NaCl)
15
 pH 7-11.
10
2
4
6
pH
8
10
12
14
Lipopeptide toxicity tests
Toxic to brine shrimp at very high concentrations
Brine shrimp
% survival
100
80
60
No brine shrimp
survive
40
20
0
Artimia assay (Luna et al., 2013)
0.5XCMC
1XCMC
0.025 %
(w/v)
0.05 %
(w/v)
2XCMC
10XCMC
50XCMC
(1:10
dilution)
0.1 % (w/v) 0.5 % (w/v) 2.5 % (w/v) dispersant
standard
Biosurfactant
No toxicity to plant seedlings
Water
Control
Biosurfactant
SDS
seawater
No toxicity to PAH-degrading bacteria
Biosurfactant
LS9TH
SDS
15
Lipopeptide based dispersant
lipopeptide conc.
• Varying from 0.25-10 % (w/v)
Seawater
Oil 20 µl
Petroleum type
• Oman light crude oil
• Arabian light crude oil
• Diesel oil
• Fuel oil
BSF
10 µl
0%
oil displacement
100 %
oil displacement
16
Lipopeptide based dispersant
Fuel Oil
Oman light crude oil
Arabian light crude oil
Diesel oil
Oil dispalcement (%)
100
80
60
40
20
0
10 (% w/v) 100 (% w/v) 1 (% w/v)
BSF foamate
3 (% w/v)
4 (% w/v)
BSF powder
10 (% w/v) 10 (% w/v) 10 (% w/v)
Slickgone NS Tween20
Oil displacement activities of foamate and powder were comparable to a commercial
dispersant (Slickgone NS) and much higher than a synthetic surfactant (Tween 20).
17
Conclusions
1
2
3
4
Lipopeptide biosurfactant could be produced from chitosan-immobilized
Bacillus sp. GY19 in stirred tank fermenter.
Lipopeptides could be recovered from cell-free culture medium by foam
fractionation process.
Lipopeptides have good surface activity, low toxicity, and stable under
various conditions.
Both foamate and powder containing lipopeptides could be used directly
as dispersants for oil spill remediation
-PTT Group Use only-
18
Acknowledgements
Sarintip Vaewhongs
Chatree Tankunakorn
Komkrit Suttiponparnit
Project advisor
Project leader
Researcher
Witchaya Rongsayamanont
Nichakorn Khondee
Onruthai Pinyakong
Ekawan Luepromchai
19