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Production and Application of Lipopeptide Biosurfactant for Dispersing Oil Spill in Seawater
Suwat Soonglerdsongpha1, Witchaya Rongsayamanont2, Nichakorn Khondee2, Onruthai Pinyakong2, Ekawan Luepromchai2
5th World Congress on Biotechnology, Valencia, SpainJune 25th -27th , 2014
1 Environmental Research and Management Department, PTT Research and Technology Institute, Thailand2 Department of Microbiology, Faculty of Science, Chulalongkorn University, Thailand
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Biosurfactant
Hydrophilic Moiety Hydrophobic Moiety
Biosurfactant
Biosurfactants are surface-active biomolecules produced by microorganisms.
• Acid• Peptide cations or anions• Mono-, di- or polysaccharides
Hydrophilic Moiety
• Unsaturated or saturated hydrocarbon chains • Fatty acids
Hydrophobic Moiety
(Surfactant head)
(Surfactant tail)
(http://www.ualberta.ca/~csps/JPPS8(2)/C.Rangel-Yagui/solubilization.htm)
CMC
(http://people.umass.edu/mcclemen/Group.html)
CMC = Critical Micelle Concentration
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Biosurfactant
Classification of Biosurfactants Glycolipids – Sophorolipids, Trehalolipids, and
Rhamnolipids Lipopeptides and Lipoproteins Fatty acids Phospholipids Neutral lipids Polymeric biosurfactants Particulate biosurfactants
Rhamnolipid Sophorolipid Surfactin (Lipopeptide)
Trehalolipid
http://biotechsupportbase.com/wp-content/uploads/2014/02/surfactants.png
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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
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Applications of Biosurfactants
Biosurfactant market volume share (by application, 2013)
Biosurfactant applications1. Household detergent
2. Personal care3. Industrial cleaner4. Food processing5. Oilfield chemicals6. Agriculture chemicals7. Textiles8. Others
(http://www.grandviewresearch.com/industry-analysis/biosurfactants-industry)
Environmental applications Soil bioremediation Oil dispersant
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Dispersant use for oil spill
Increase natural attenuation and biodegradation by microorganism.
Blend of two or three surfactants Ex. nonionic /anionic/solvent
Oil droplets
However, synthetic dispersants are usually toxic and may decrease biological activity of microorganisms.
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Objectives
Production & downstream processes
Microorganisms Biosurfactants
Substrates
Low cost substrates???
Biosurfactant producing Microorganisms??? Production & downstream
Processes???
Dispersing oilapplication???
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Scope of Experiment
Isolation of biosurfactant-producing bacteria
Evaluation of substrate utilization
Biosurfactant
Production of biosurfactant by chitosan-immobilized cell
Purification of biosurfactant by Foam fractionation and Freeze-drying
Characterization of biosurfactant
Properties & stabilities of biosurfactant
Application of biosurfactant for dispersing oil
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Biosurfactant producing microorganisms & Substrate selection
-PTT Group Use only-
ดิ�น ทราย ดิ�นป่าชายเลน ใบไม้�
น��าทะเล น��าคลอง น��าเสี�ย คล�งเก็�บจุ�ล�นทร�ย�
Pure substrate
Organic waste
Glycerol Soybean oil Palm oil Crude oil Diesel Lube
Slop oil Bottom glycerol
High yield Low cost Waste reduction
58 species
107species
Surface tension
< 40 mN/m
Bacillus sp. GY19
Highest yield
Microbial screening
Isolation of microorganisms
Substrate Selection
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Screening of biosurfactant-producing bacteria
Surface tension
< 40 mN/m
58 strains 20
genus
Bacillus sp. GY19
Yield
0.13
g/l
Pure glycero
l
NH
NH
HN
O CH3
CH3
O
CH3
CH3NHO
HO
O
OOO
NHH3C
CH3O
HN
CH3
H3CO
HN
CO2H
O CH3
CH3
Val
Asp
Glu
Leu
Leu
Leu
Leu
myristoleic acid
BSF PGBiosurfactant from pure glycerol
Surfactin
Bottom glycerol + palm
oilYield 2.8 g/l
Surfactin
Lipopeptide biosurfactant
107 strains
Highest yield
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Lipopeptide production and recovery processes
Low cost substrate
High production yield
Foam fractionation Solvent-free method
Waste utilization
Immobilized bacteria on cheap and effective
materials
Efficient recovery and purification methods
Foamate easy to use and effective for further
applicationDevelop
Cheap, easy and effective bioprocess
Chitosan-immobilized cells Foam fractionation
Bottom glycerol(10% glycerol + fatty
acids and etc.)
Substrate Production Downstream
Waste from biodiesel production
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Lipopeptide production by chitosan immobilized cells in stirred tank bioreactor
- Palm oil was added as precursor
- Maximum lipopeptide yield 6.65 g/L
0 5 10 15 20 250123456789
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0
20
40
60
80Crude biosurfactant Oil Glycerol
Time (day)
Crud
e bi
osur
fact
ant (
g/L)
Rem
aing
ing
oil (
g/L)
Rem
aini
ng g
lyce
rol (
g/L)
Cycle3
Cycle2
Cycle1
Cycle4
Cycle5
Cycle6
A
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Properties of lipopeptide biosurfactant
Foam-fractionati
on
Foamate
Cell free supernatant Biosurfactant powder 50% lipopeptide (w/w)
Freeze-drying
0.001 0.01 0.1 1 100
10
20
30
40
50
60
70
1st batch production
2nd batch production
3rd batch production
Biosurfactant powder conc. (%, w/v))
Su
rfac
e te
nsi
on
(m
N/m
)
Concentration of foamate (%)
Lowest SFT ~ 26 mN/m
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Stabilities of lipopeptide biosurfactant
30 40 50 60 70 8010
15
20
25
30
35
40
Temperature (C)
surf
ace
tens
ion
(mN
/m)
2 4 6 8 10 1210
15
20
25
30
35
40
pH
surf
ace
tens
ion
(mN
/m)
0 1 2 3 4 5 6 7 8 9 1010
15
20
25
30
35
40
NaCl (%)
surf
ace
tens
ion
(mN
/m)
Surface activity was stable at
Temperature (40-100oC)
Electrolytes (> 6% NaCl)
pH 7-11.
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Lipopeptide toxicity tests
0.5
XC
MC
1XC
MC
2XC
MC
10X
CMC
50X
CMC
(1:1
0 d
iluti
on)
0.025 % (w/v)
0.05 % (w/v)
0.1 % (w/v)
0.5 % (w/v)
2.5 % (w/v)
dispersant standard
Biosurfactant seawater
0
20
40
60
80
100
% s
urv
ival
No brine shrimp survive
Brine shrimp
Artimia assay (Luna et al., 2013)
Biosurfactant
LS9TH SDS
No toxicity to PAH-degrading bacteria
Water
Biosurfactant
SDS
No toxicity to plant seedlings
Toxic to brine shrimp at very high concentrations
Control
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Lipopeptide based dispersant
Seawater Oil 20 µl BSF10 µl
100 % oil displacement
0 % oil displacement
lipopeptide conc.
• Varying from 0.25-10 % (w/v)
Petroleum type
• Oman light crude oil• Arabian light crude oil• Diesel oil• Fuel oil
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Lipopeptide based dispersant
10 (% w/v) 100 (% w/v) 1 (% w/v) 3 (% w/v) 4 (% w/v) 10 (% w/v) 10 (% w/v) 10 (% w/v)BSF foamate BSF powder Slickgone
NSTween20
0
20
40
60
80
100
Fuel Oil Oman light crude oil Arabian light crude oil Diesel oil
Oil
disp
alce
men
t (%
)
Oil displacement activities of foamate and powder were comparable to a commercial dispersant (Slickgone NS) and much higher than a synthetic surfactant (Tween 20).
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Conclusions
-PTT Group Use only-
2
3
4
Lipopeptide biosurfactant could be produced from chitosan-immobilized
Bacillus sp. GY19 in stirred tank fermenter.1
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
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Acknowledgements
Sarintip Vaewhongs Project advisorChatree Tankunakorn Project leaderKomkrit Suttiponparnit Researcher
Witchaya RongsayamanontNichakorn KhondeeOnruthai PinyakongEkawan Luepromchai