1

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

2

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

3

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

4

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

5

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

6

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.

7

Objectives

Production & downstream processes

Microorganisms Biosurfactants

Substrates

Low cost substrates???

Biosurfactant producing Microorganisms??? Production & downstream

Processes???

Dispersing oilapplication???

8

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

9

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

10

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

11

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

12

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

10

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

13

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

14

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.

15

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

16

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

 

17

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).

18

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

19

Acknowledgements

Sarintip Vaewhongs Project advisorChatree Tankunakorn Project leaderKomkrit Suttiponparnit Researcher

Witchaya RongsayamanontNichakorn KhondeeOnruthai PinyakongEkawan Luepromchai