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Production of a Peptidoglycolipid Bioemulsifier by Pseudomonas aeruginosa
Grown on Hydrocarbon
Matthew O. Ilori* and Dan-Israel Amund
Department o f Botany and Microbiology, Univ er sity of Lagos, Akoka - Yaba, Lagos, Nigeria
E-mail: olusojiilori@ yahoo.com
* Author for correspo ndence and reprint req uests
Z. Naturforsch. 56c, 547-5 52 (2001); received February 6/March 8, 2001
Pseud omon as, Hydrocarbon, Peptid oglycolipid
A strain o f Pseudomon as aeruginosa isolated from a polluted soil was fo und to produce
an extracellular bioemulsifier w hen cultiva ted on hexad ecan e as sole carbon source. The
emulsifier was precipitated with a ceton e and red issolved in sterile water. Dod ecane , crude
oil and kerosene were found to be go od substrates for emulsifica tion by the bioemulsifier.
Growth and bioemulsifier production reached the optimal lev els on the fourth and fifth day,
respectively. Emulsifying activity was observed over a pH range o f 3.5 to 10.0 with a maxi
mum at pH 7.0. The activity o f the bioemulsifier was heat stab le up to 70 °C while about 50
percent o f its activity was retained at 100 °C. The com pon ents o f the bioem ulsifier were
determined, it was found to contain carbohydrate, protein and lipid. The p ro tein comp lex
was precipitated with ammonium sulphate and fractionated on a Sep hadex G-100. Gel ele c
trophoresis of the bioem ulsifier show ed a single band whose molecular w eight was estim ated
as 14,322 Da. The bioem ulsifier was classified as a pep tido glycolipid. Certain strains of P.
aeruginosa produce peptid oglyco lipid in place of rhamnolipid.
Introduction
The growth of microorganisms on hydrocarbons
is often accompanied by the emulsification of the
insoluble carbon source in the culture medium
(Rosenberg et al., 1979). The spontan eous release
and function of biosurfactants are often related to
hy dro carbon uptake. Therefore, they are pre dom i
nantly synthesized by hydrocarbon-degrading bac
teria and fungi. Biosurfactants are a structurally
diverse group of surface active molecules synth e
sized by microorganisms which can be accum u
lated as cell bound or extracellular products. Many
hydrocarbon utilizing bacteria and fungi have
been reported to possess emulsifying activities due
in part to the whole cells or to extracellular sur
face-active compounds. Emulsifying ability, after
removal of cells, often disappear in cells participat
ing in emulsification whereas isolates secreting
surface active compounds extracellularly often re
tain emulsifying ability after removal of cells. Ex
tracellularly produced surface active com pounds
are the most promising biosurfactants. Some of the
compounds with emulsifying properties include
phospholipids, fatty acids, lipopeptides, lipopro
teins and glycolipids. Polysaccharides have no
emulsification activity alone but may become po
tent emulsifiers when combined with proteins or
lipids released during growth (Desai and Banat,
1997). Biosurfactants can be divided into two m a
jo r categories. The first are the low molecular
weight surfa ctants such as glycolipids, fatty acids
and phospholipids. The second type are the high
molecular weight polym ers (Willumsen and Karl-
son, 1997). M ost known biosurfactants are glyco
lipids. Amon g the glycolipids, the best known are
rhamnolipids, trehalolipids. Rhamnolipids, pro
duced by P. aeruginosa are the best studied and
the most reported glycolipids and was first de
scribed by Jarvis an d Johnson (1949). Several
other au thors including Hisatsuka et al. (1971),
Itoh and Suzuki (1972), Reiling et al. (1986) and
Ochsner et al. (1995) have described the rhamnoli
pids of P. aeruginosa. Polymeric emulsifiers in
clude emulsan, liposan, alasan, man noprote in and
other polysaccharide protein complexes (Desai
and B anat, 1997). Emulsan is a polyanionic amphi-
pathic heteropolysaccha ride bioemulsifier pro
duced by A cine toba c ter calcoaceticus RA G - 1.
Liposan is an extracellular emulsifier produced by
Ca ndida lipolytic a, it is composed of carbohydrate
and p rotein with the carbohydrate p ortion con
sisting of glucose, galactose, galactosamine and ga-
0939-5075/20 01/0 70 0-05 47 $ 06.00 © 2001 Verlag der Zeitschrift für Naturforschung, Tübingen • www.znaturforsch.com ■ D
548 M. O. Ilori and D. I. Amund • Bioemulsifier from P. aeruginosa
lacturonic acid. A lasan is a heterop olysacc har
ide - protein biosurfactant produced by
Ac ineto bacter radioresistens . M annoprote in is
prod uced by S accharom yces cerev isiae, the protein
has been reported to have excellent emulsifying
activity towards several oils, alkanes and organic
solvents (Desai and Banat, 1997). Few studies
have, however, emerged on the polymeric bioe
mulsifier of P. aeruginosa such as peptidoglycoli-
pid.
In recent years, interest in the isolation and
identification of new microbial bioemulsifiers has
increased immensely due to their ability to meet
most synthetic surfactants’ pro p ertie s including
emulsification, wetting, phase separation and vis
cosity reduction for potential applicatio n in the
cosmetics, food and agricultural industries. In this
paper, we re port some properties of a peptidogly-
colipid bioemulsifier produced by an isolate of P.
aeruginosa.
Materials and Methods
Bacterial strain
The organism used in this study was isolated
from a Nigerian soil polluted with crude oil using
minim al salts agar as described by West et al.
(1984). The isolate was identified on the basis of
Gram reaction, cell morphology and other bio
chemical tests as described by H olt et al. (1994).
Growth conditions
The m edium for the production of bioemulsifier
contained per litre: N H4N03, 4.0 g; N a2HP04, 2.0 g;
KH2P04, 0.53 g; K2S04, 0.17 g, MgS04. 7H20, 0.10 g
and n-hexadecane (1.0% w/v) as sole carbon and
energy source. The pH of the medium was ad
justed to 7.1 before autoclaving at 121 °C for
15 min. Trace elements solution (2.5 ml per litre)
described by Bauchop and Elsden (1960) was s ter
ilized separately and then added aseptically to the
medium. The medium was inoculated with the or
ganism and incubated with shaking (120 rev/min)
at 30 °C.
Growth o f organism , bioem u lsifier p rodu ction
an d p urificatio n
Studies on growth of the organism and bioemul
sifier production over a time course was carried
out. Samples were aseptically withdrawn, diluted
in sterile distilled water and aliquots plated in trip
licate on nutrient agar for total viable counts. The
cells were removed from the culture broth by
centrifugation (10,000xg, 15 min, 4 °C) while the
clear supern atan t was used as the source of crude
bioemulsifier.
The bioem ulsifier was recovered from the cell
free culture supernatant by acetone precipitation.
Three volumes of chilled acetone was added and
allowed to stand for 10 h at 4 °C. The precipitate
was collected by centrifugation and evap orated to
dryness to remove residual acetone after which it
was redissolved in sterile water.
Em u lsification measurem ent
The activity of the purified bioemulsifier was
measured twice in all cases by adding 2.5 ml of n-
dodecane to 2.5 ml of bioemulsifier in a test-tube.
The tube was vortexed at high speed for 2 min.
and left undistu rbed for 24 h. M easurem ents were
made manually th ereafte r to get the average em ul
sion index. The emulsion index (E24) was the
height of the emulsion layer divided by the total
height multiplied by 100. Hexadecane, octane, ker
osene, crude oil and hexane were also assayed for
their ability to serve as substrates for emulsifica
tion.
Effect o f pH and temperature;
protein determination
The pH of the purified bioemulsifier was ad
justed to a range of 3.5-10.0 with dilute HC1 or
NaOH after which its activity was determ ined.
Samples of the bioemulsifier were also exposed to
heat for 30 min at a temperature range of 27 -
121 °C and its activity determ ined thereafter.
The p rotein content of the purified bioemulsi
fier was assayed by the Bradford (1976) m ethod
with bovine serum albumin as standard.
Ca rboh ydra te and lipid analysis
The carbohy dra te conten t of the purified bio
emulsifier was determined as total hexoses by the
M. O. Ilori and D. I. Amund • Bioem ulsifie r from P. aeru ginosa 549
anthrone reagent (Spiro, 1966). To the sample
(1.0 ml) was a dded 5.0 ml of cold anthrone reagent
in a test tube. The tubes were shaken vigorously
to ensure mixing and h eated in a boiling water
bath for 15 min at the end of which the tubes were
cooled in a water bath and allowed to stand for
20 min followed by absorbance m easurement at
620 nm. The blank contained 1.0 ml of distilled
water and 5.0 ml of the anth rone reagent. The car
bohydrate c onten t in samples were extra polated
from a standard calibration curve prepared with
known concentrations of glucose.
Thin layer chrom atography (TLC) was carried
out on a 20 x 20 cm precoated silica gel plate using
petroleum ether, diethyl ether and acetic acid
(90:10:1) as running solvent. The plate was devel
op ed by staining with 5% H 2S04 in 95% ethanol
followed by heatin g at 150 °C for 30 min. The Rf
value of the developed spot was calculated and
com pared with values of standard compounds in
sim ilar solvent as described by Kates (1972).
Protein purification and m olecu lar weight
determ ination
The pro tein complex of the bioemulsifier was
precipitated with (NH4)2S 0 4 (analytical grade) at
80% s aturation. The precipita te was collected by
centrifugation, redissolved in sodium phosphate
buffer (0.05 m, pH 7.0) and dialyzed against the
buffer at 4 °C for 12 h. The dialyzed sample was
fractionated on a Sephadex G-100 column as de
scribed by Ilori et al. (1995). The protein content
of fractions was determined after Bradford (1976)
while the apparent molecular weight was esti
mated using sodium dodecyl sulfate - polyacryl
amide gel electrophoresis (SDS-PAGE, 30%). The
gel was stain ed with Coomassie blue reagent. The
following m arker proteins (Sigma, U SA) were
used: bovine serum albumin (66,000 Da), egg albu
min (45,000 D a), glyceraldehyde-3-phosphate-de-
hydrogenase (36,000 Da) bovine carbonic anhy-
drase (29,000 Da), bovine pancreas trypsinogen
(24,000 Da), soybean trypsin inhibitor (20,000 Da)
and bovine milk a-lactalbum in (14,200 Da).
The hot phenol method described by Navon -
Venezia et al. (1995) was used to ascertain whether
or not the pro tein moiety was essential for emulsi
fying activity.
Results
The microbial strain used in this study, Pseu-
do m ona s aeruginosa, was an anthracen e deg rad er
isolated from soil polluted with crude mineral oil.
The organism was rod shaped, Gram negative, cat-
alase positive, oxidase positive, m otile and did not
produce indole. It however produced pyocyanin
and pyoverdin pigments. The organism grew at
42 °C and utilized glucose, raffinose, sucrose, man-
nitol and maltose but did not utilise trehalose and
sorbitol as sources of carbon. The organism grew
in minimal salts medium containing n-hexadecane
as sole carbon source and secreted extracellular
compounds with surface active properties. The
growth of the organism on n-hexadecane was ac
companied by an increase in viable cell counts.
Maximum production o f the bioemulsifier oc
curred on the 5th day (Fig. 1A). A decrease in the
viable cell counts was observed on the 5th and 6th
day.
Emulsifying activity was observed over the en
tire pH range studied. The purified bioemulsifier
was very active at p H 6 and 7 and fairly active at
pH 4 -5 and 8 -9 . Low emulsifying activity was
however observed at p H 3.5 and 10 (Fig. IB). The
activity of the purified bioemulsifier was stable up
to 70 °C after which a sharp dro p in activity oc
curred (Fig. 1C). At 100 °C, abo ut 50% of em ulsi
fying activity was reta ined.
The ability of a range of selected liquid hy
droc arbons to serve as substrates for emulsifica-
tion was investigated. The results presented in
Fig. 2 show ed that n-dodecane was the best sub
strate emulsified. C rude oil and kerosene also
served as good substrates for emulsification. The
least emulsified substrate was n-hexane. Hexade-
cane which was the substrate used in cultivation of
the cells was fairly emulsified. Em ulsion formed
with kerosene, crude oil, and dodecane were sta
ble for at least 48h. The emulsion form ed with
crude oil separated when allowed to stand undis
turbe d for m ore th an 48h into an upper turbid
emulsion and a lower organic phase. On agitation
the two phases mixed immediately forming a
cream y emulsion.
The purified bioemulsifier of P. aeruginosa had
protein and carbohy drate components of approxi
mately 28% and 34% respectively. A single SDS -
PAGE band was obtained from the purified pro-
550 M. O. Ilori and D. I. Amund • Bioem ulsifier from P. aerug in osa
lane 1 lane 2
2 3 4 5
Tim e ( Da ys )
14,322 Da
66,000 Da
20,000 Da
14,200 Da
Fig. 3. Polyacrylamide gel electrophoresis.
Lane 1, 10 |_il of purified b ioem ulsifier produced by P.
aeruginosa,
Lane 2, 10 [.il o f standards: bovine albumin (M r -
66.000), egg albumin (M r = 45,000), glycer aldehyde-3-
phosph ate-dehydrogenase (M r = 36,000), bovine car
bonic anhydrase (M r = 29,000), bovine Pancreas trypsin-
ogen (M r = 24,000), Soybean trypsin inhibitor (M r =
20.000) and bovin e milk a-lactalbu min (Mr = 14,200).
Temp . ( °C )
Fig. 1A. Time course production of bioemulsifier by P.
aeruginosa A , bioem ulsifier; Log. number of viable
cells. Total viable cells (T V C) ranged from 9.3 x 105 to
6.03 x 109.
B. Effect of pH on bioemulsifier production by P. aeru gi
nosa.
C. Effect of tem perature on bioem ulsifier prod uction by
P. aeruginosa.
100% value = emu lsion index (E 24) 70.
Su b st ra tes
Fig. 2. Substrate specificity of the bioemulsifier.
tein complex preparation (Fig. 3, lane 1). The ap
paren t molecular weight was estimated as 14,322
Da.
After hot phenol treatment, no emulsifying ac
tivity was found in the aqueous deproteinized frac
tion. This indicated that the emulsifying activity
was totally associated with the denatured protein
fraction. The analysis of the purified bioem ulsifier
by TLC showed a single spot of lipid that was not
identified. The bioemulsifier was subsequently
classified as a peptidoglycolipid.
Discussion
Biosurfactants are a heterogenous group of su r
face - active molecules synthesized by m icroorga
nisms such as bacteria, yeasts and moulds. Biosur
factant producers among the bacteria include
Ac ineto bacter sp, Pseudomona s sp. Bacillus sp and
Corynebacteriu m sp. A soil isolate of Ps eu d om o
nas aeruginosa used in this work grew on n-hex-
adecane and produced bioemulsifier. The p ro
duction of the bioemulsifier was growth
associated. Grow th associated production of sur
face active compounds have been reported from
several other microorganisms such as Bacillus
stearothermophilus VR-8 (G urjar et al., 1995),
Corynebacterium lepus (Duvnjak and Kosaric,
1985), Candida lipolytica (Cirigliano and Carman,
M. O. Ilori and D. I. Amu nd • Bioemu lsifier from P. aeruginosa 551
1984) and Arthro b acter RAG-1 (R osenberg et al.,
1979).
Most microbial emulsifiers are substrate spe
cific, solubilizing or emulsifying different hy
drocarbon s at different rates. Dodecane, crude oil
and kerosene served as good substrates for emulsi
fication by the peptidoglycolipid of P. aeruginosa.
The emulsification of crude oil by the peptidogly
colipid was not surprising because crude oil is a
mixture of hydrocarbons and would naturally p r e
sent the emulsifier with substrates of choice.
Em ulsification of crude oil by the peptidoglycoli
pid makes it suitable for oil and storage tank
clean up.
The peptidoglycolipid was most active at pH 7.0,
very active at pH 6.0 and fairly active at acidic and
alkaline pH. Different emulsifiers are known to
have different pH optim a. The optimum pH for
the emulsifier of Acinetoba cter radioresistens was
re p o rted to be 5.0 (N avon - Venezia et al., 1995).
The emulsifier of Bacillus cereus was only active
at pH below 7.0 while at a pH above 7.0, a p ro
no unced loss of activity was recorded (Cooper and
Golden berg, 1987). The wider pH range of activity
of the peptidoglycolipid of P. aeruginosa makes it
more suitable for use in soil and aquatic environ
ment b ioremediation relative to those with n ar
rower pH range. The peptidoglycolipid was heat
stable and retained about 50% of its activity at
100 °C. In term s of h eat stability, the peptidogly
colipid of P. aeruginosa appeared better than lipo
san prod uced by Candida lipolytica reported to be
stable at tem p eratures up to 70 °C and with about
60% loss in activity afte r heating at 100 °C (Cirig-
liano and Carman, 1984).
The peptidoglycolipid had a protein activator
that was involved in emulsification. Hisatsuka et
al. (1972, 1977) described the isolation from P. aer
uginosa of a protein activator th at was involved in
emulsification of hydrocarbons, it had a m olecular
weight of 14,300 and contain ed 147 amino acids of
which 51 were serine and threonine. The protein
com ponent of a bioemulsifier produced by P. aeru
ginosa P-20 was repo rted to bear 52 amino acids
(Koronelli et al., 1983). The molecular weight of
the em ulsifying proteins p rodu ced by P. cepacia
N1 was estimated as 14,000 D a (Goswami et al.,
1994). In term s of molecular weight, the protein
com plex produced by P. aeru ginosa reported in
this work appeared closely sim ilar to that de
scribed in P. aeruginosa by Hisatsuka et al. (1972).
The bioemulsifier had a lipid com ponent that was
not identified. Some known lipids rep o rted to be
involved in bioemulsification include monoglycer
ides, 1,2-diglyceride and corynomycolic acids
(C ooper and G oldenberg, 1987). Lipids improve
emulsion stability by reducing interfacial tension
betw een two immiscible phases.
The peptidodglycolipid reported in this work
was found to be different from rhamnolipid pro
duced by m any strains of P. aerugin osa. The broad
pH and tem perature stability of this bioemulsifier
indicate its potential for exploitation in areas such
as soil an d w a ter clean up, cosm etic and food indu
stries.
Ac knowledgem ents
Dr. M. O. Ilori gratefully acknowledges U N
ESCO for the award of a short-term fellowship in
Biotechnology at the University of Kent at Can
terbury, U. K. w here this work was carried out and
thank Professor Alan Bull, Dr. Gary Robinson
and Dr. Steve H e ald of the D epartm ent of Biosci
ences, University of Kent at C anterbury, U. K. for
technical support.
552 M. O. Ilori and D. I. Amund • Bioem ulsifier from P. aeruginosa
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