ORIGINAL ARTICLE

Production of rhamnolipids with a high specificityby Pseudomonas aeruginosa M408 isolatedfrom petroleum-contaminated soil using olive oil as sole carbonsource

Fangling Ji1 & Lu Li1 & Shuai Ma1 & Jingyun Wang1 & Yongming Bao1

Received: 28 July 2015 /Accepted: 2 February 2016 /Published online: 16 February 2016# Springer-Verlag Berlin Heidelberg and the University of Milan 2016

Abstract Rhamnolipids (RLs) represent a very promisingclass of biosurfactants that have many applications as biocom-patible and biodegradable emulsifiers. We have isolated aPseudomonas aeruginosa strain, denoted strain M408, frompetroleum-contaminated soil of the Liaohe oil field (northernChina) that is able to produce RLs with a high specificitywhen grown on olive oil as the sole carbon source. Productanalysis of these RLs showed that only di-RLs (F1 sample),including rhamnose (Rha)-Rha-C10-C10 and Rha-Rha-C10-C12/Rha-Rha-C12-C10, and a single mono-RL (F2 sample)of Rha-C10-C10 were produced by the isolate. Further testingrevealed that these F1 and F2 products were able to reduce thesurface tension of water to 31 and 27 mN/m, respectively. Thecritical micelle concentration was 120 and 60 mg/L for the F1and F2 products, respectively. The F1 and F2 products alsoshowed good emulsion stability, achieving an emulsion capac-ity of 87 and 90 %, respectively, with atoleine after 24 h at alow concentration of 100 mg/L. Maximum production of theRLs was obtained after optimization of the culture conditions,with a 6.85-fold increase in the yield of the RLs, up to12.6 g/L, relative to the yield before optimization.

Keywords Pseudomonas aeruginosa . Rhamnolipid .

Biosurfactants . Olive oil

Introduction

Biosurfactants are extracellular, surface-active, amphipathicmolecules produced by a wide variety of microorganisms.They are highly diverse in terms of chemical structure, phys-ical properties and composition and include peptides, glyco-lipids, lipopeptides, fatty acids and phospholipids (Rahmanand Gakpe 2008). When compared with chemical syntheticsurfactants, biosurfactants have a low toxicity, are biodegrad-able and show high selectivity and specificity even at extremepH, temperature and salinity conditions. These features makebiosurfactants Bgreener^ alternatives to chemical surfactantsand potential candidates for a wide range of applications, in-cluding environmental remediation (Kiran et al. 2010), anti-microbial agents (Kryachko et al. 2013), food industry(Khopade et al. 2012), oil-recovery processes (Ławniczaket al. 2013), nanotechnology (Sachdev and Cameotra 2013),among others.

Glycolipids are the most common type of biosurfactant andcontain both a fatty acid hydrophobic moiety and a hydrophil-ic carbohydrate component (Müller and Hausmann 2011).Rhamnolipid (RL) biosurfactants, which are produced by bac-teria of the genus Pseudomonas, are the most studied speciesof glycolipid biosurfactants (Henkel et al. 2012). They arecomposed of one (i.e. mono-RLs) or two (i.e. di-RLs) rham-nose sugar moieties linked to one or two β-hydroxy fatty acidchains. To date, at least 28 different structural homologs, allproduced by Pseudomonas strains grown on different carbonsources, have been reported in the literature (Monteiro et al.2007; Dobler et al. 2016). The production of RLs byP. aeruginosa has been widely reported (Pajarron et al.1993; Thanomsub et al. 2007; Kumar et al. 2012), with the10-carbon molecule chains found to be the predominant struc-tural form of RLs synthesized by P. aeruginosa (Déziel et al.2000). Other minor rhamnolipidic homologs involving C8,

Fangling Ji and Lu Li contributed equally to this work.

* Fangling Jifanglingji@dlut.edu.cn

1 School of Life Science and Biotechnology, Dalian University ofTechnology, No. 2 Linggong Road, Ganjingzi District,Dalian 116024, China

Ann Microbiol (2016) 66:1145–1156DOI 10.1007/s13213-016-1203-9

C10, C12 and C14 3-hydroxy fatty acids have also been ob-served (Déziel et al. 1999; Mata-Sandoval et al. 1999; Dézielet al. 2000).

Although the advantages of biosurfactants over chemi-cal surfactants have been demonstrated in many studies,the application of these compounds is still not widespreaddue to the relatively high production costs (Gudina et al.2015). The utilization of renewable raw substrates and opti-mization of medium composition have been described as twostrategies which enhance the production of biosurfactants(Soberón-Chávez and Maier 2011). The dependence of RLproduction on the composition of the cultivation mediumhas been recognized in many studies (Mehdi et al. 2011;Henkel et al. 2012; Rikalovic et al. 2012). More specifically,the carbon (C) and nitrogen (N) source and the C/N ratio (Liet al. 2011), as well as the presence and concentration of traceelements, especially divalent cations (Mehdi et al. 2011), havebeen found to have a significant impact on the amount andcomposition of the RLs produced (Li et al. 2011).

In this framework, we have isolated an indigenous bacterialstrain capable of producing biosurfactants from petroleum-contaminated soil of a local oilfield. The strain, identified asPseudomonas aeruginosaM408, was evaluated for its poten-tial to produce biosurfactants. The RLs in the supernatant ofP. aeruginosa M408 cultures were purified and identified bygas chromatography–mass spectrometry (GC-MS) and massspectrometry electrospray ionization–tandem mass spectrom-etry (ESI-MS/MS). The surface activity of the RLs producedby P. aeruginosa M408 was analyzed in terms of surfacetension reduction and emulsification activity for substrates.The isolated strain was cultivated in different media and underdifferent culture conditions to determine the optimal culturestrategy for the production of the RLs. Different carbonsources, including olive oil, soybean oil, atoleine, glycerol,glucose and sucrose, were tested to explore their effects onthe yield of RLs. The objective of this work was to assess thepotential use of P. aeruginosa M408 for the industrial-scaleproduction of RLs.

Materials and methods

Materials and media

All the chemicals and reagents used in the study were fromcommercial sources and of the highest purity available.

Enrichment medium (g/L): olive oil, 20; (NH4)2SO4, 10;KH2PO4, 3.4; K2HPO4 · 3H2O, 4.4; MgSO4 · 7H2O, 1;NaCl, 1.1; KCl, 1.1.Oil plates (g/L): NaNO3, 5; KH2PO4, 3.4; K2HPO4 ·3H2O, 4.4; MgSO4 · 7H2O, 1; NaCl, 1.1; KCl, 1.1, agar,

20. A layer of sterilized olive oil was coated on the sur-face of the solidified medium.Seed medium (g/L): yeast extract, 5; peptone, 10; NaCl,10; pH 7.0.Fermentation medium: carbon (olive oil, soybean oil, liq-uid paraffin, glycerin, glucose and sucrose) and nitrogen((NH4)2SO4, NH4NO3, NaNO3, peptone and urea)sources as specified; other components (in g/L):KH2PO4, 3.4; K2HPO4 · 3H2O, 4.4; MgSO4 · 7H2O, 1;NaCl, 1.1; KCl, 1.1; pH 6–10.

Isolation, screening and identificationof biosurfactant-producing microorganism

Pseudomonas aeruginosa M408 was isolated from soilsamples collected at the Liaohe Oilfield in China. A 2 gpetroleum-contaminated soil sample was initially cultivatedin enrichment medium supplemented with olive oil as the solecarbon source for a period of 4 days. These enriched bacteriawere then serially diluted and plated onto oil plates overlaidwith olive oil, which acted as the sole carbon source, and theplates incubated at 28 °C for 72 h; the colonies which ap-peared were picked and further purified on nutrient agar plates(3 g/L beef extract, 10 g/L tryptone, 5 g/L NaCl, 20 g/L agar,pH 7.0) by restreaking two to three times to obtain singleisolated colonies. These isolates were maintained on nutrientagar slants and stored at 4 °C, and also as stocks in glycerol(25 %, v/v), stored at −80 °C.

The bacterial isolates were further screened forbiosurfactant production by the oil spreading method(Bodour and Miller-Maier 1998) and Wilhelmy plate methodfrom the cell-free culture and the crude extract. The strainwhich presented with the widest clear zone using the oilspreading technique and the lowest surface tension was sub-sequently characterized.

The extracted genome DNA of the isolated bacterium(using the TaKaRa MiniBEST Bacteria Genomic DNAExtraction kit; Takara, Tokyo, Japan) served as the templatefor 16S rDNA gene amplification (using the TaKaRa 16SrDNABacterial Identification PCR kit; TaKaRa). The forwardprimer (5′-ACG CTG GCG GCA GGC CTA ACA CATGCA-3′) and the reverse primer (5′-ACC ACG GAG TGATTC ATG ACT GGG-3′) were used for 16S rRNA gene am-plification. This set of primers was designed based on thebeginning and end conservative regions of the 16S rDNA toamplify the entire approximately 1.5-kbp 16S rRNA gene.The primers used for sequencing were a forward primer(5′-GAGCGGATAACAATTTCACACAGG-3′), a reverseprimer (5′-CGC CAG GGT TTT CCC AGT CAC GAC-3′)and an internal primer (5′-CAG CAG CCG CGG TAATAC-3′). The PCR cycling conditions included a denaturation stepat 94 °C for 5 min, followed by 30 cycles of 60 s at 94 °C, 60 s

1146 Ann Microbiol (2016) 66:1145–1156

at 55 °C and 90 s at 72 °C, with a final primer extension step of5 min at 72 °C. Amplified PCR products were purified usingthe TaKaRa Agarose Gel DNA Purification kit ver.2.0(TaKaRa). Sequencing of the purified PCR products wereperformed by TaKaRa China (Dalian, China). The Genbankaccession number (HQ222816) of the 16S rDNA sequence ofthe isolate strain has been deposited in the National Center forBiotechnology Information (NCBI) database.

Isolation, purification and characterization of RLs

Rhamnolipids recovered from the crude glycolipid productharvested after 2 days of culture were isolated, purified andcharacterized. Cells were first removed from the culture bycentrifugation at 8000 rpm/min for 10 min at 20 °C. Thesupernatant was added to a 3-L Erlenmeyer flask and mixedwith an equal volume of CH3Cl:CH3OH (2:1, v/v) mixture,with shaking for 2 min. The lower CH3Cl phase was thenremoved and dried in a rotary evaporator at 40 °C. About2 g of crude extracted mixture was re-dissolved in 5 mLCH3Cl and subjected to chromatography on 50 g activatedsilica gel (approx. 60–100 mesh). The loaded column(50×3 cm) was rinsed with CH3Cl at a flow rate of 1 mL/min until neutral lipids were totally eluted. Each 20-mL ali-quot was fractionated and detected by thin-layer chromatog-raphy (TLC). The mobile phases CHCl3:CH3OH (180:25,v/v) were used to primarily elute the first homologous species(F2), and then pure methanol was used to elute the other spe-cies (F1), until all RLs had been completely recovered, asdetermined by TLC analysis. All fractions containing F1 orF2 RL species were respectively combined and concentratedusing a rotary evaporator (RE-52A; Yarong, Shanghai, China)and then quantified by weighing after extensive desiccationunder high vacuum at 40 °C.

TLC was carried out on a 60 GF-254 silica gel plate on analuminum sheet (20×20 cm; Merck, Darmstadt, Germany)using CHCl3/CH3OH/CH3COOH (180:25:1, v/v/v) as sol-vent. A quantity of 0.3 g of partially purified lipids was dis-solved in 1 mL of methanol, and 25 μL of the sample wasuniformly spotted onto the TLC sheet using a micropipettetube. The total sugar content was determined using the phenolsulfate method. The lateral edges of the sheets were firstsprayed with phenol:sulfuric acid:ethanol (3:5:95, w/v/v)and then developed at 110 °C for 5 min to stain the spots.For detection of amino acids, amines and amino sugars, theplate was also sprayed with 0.5 % ninhydrin dissolved in95 mL butanol and 5 mL 10 % AcOH. The plate was sprayedwith a 0.2 % solution of 2′,7′-dichlorofluorescein in 95 %ethanol and then dried with warm air before being viewedunder 360 nm UV light to elucidate UV-active spots contain-ing lipid components. Positive spots in corresponding bandsof four sheets, partitioned on the basis of the size of the col-ored area after treatment with the visualization reagent, were

scraped off, collected and then extracted with 4 mL of CHCl3/CH3OH (2:1, v/v).

Analysis of monosaccharides in the RL mixtures was per-formed by GC on an Agilent/HP 6890 series gas chromato-graph (Agilent Technologies, Santa Clara, CA) equipped witha HP-5 ms column (30 m×0.32 mm; 0.25 μm) using theelectron impact ionization mode. Before the mixture wereinjected into GC-MS system, the purified glycolipids weredissolved in 1 M trifluoroacetic acid, incubated at 100 °C for8 h, filtered and evaporated in a rotary evaporator. The residuewas dissolved in 1 mL of methanol and then subjected to TLCusing the standard sugar samples as controls. The TLC solventwas CH3(CH2)3OH/CH3CH2OH/H2O (4:1:2, v/v/v) and thedeveloper was phenol sulfate. Following TLC separation,1 mg of each separated monomer was dissolved in 0.5 mLof 60 g/L hydroxylamine hydrochloride–pyridine solution.The sealed solution was then allowed to react for 30 min at90 °C in an oil bath, following which 0.5 mL acetic anhydridewas added to the cooled reaction mixture and the solution wasallowed again to react for 30 min at 90 °C in an oil bath. Thefinal product was analyzed by GC under the following condi-tions: nitrogen flow of 1 mL/min, oven temperature of 60 °Cfor 11 min, followed by a ramp of 15 °C/min to 180 °C and afinal hold at 180 °C for 2 min. Sample injections were insplitless mode at 270 °C. The detector temperature was setat 280 °C.

The fatty acid composition of the RL mixtures was deter-mined by GC-MS (HP 6890 GC/5973 MS; Hewlett–Packart,Palo Alto, CA). Five milligram of RL mixture was dissolvedin 1 mL solution of 0.5 mol/L sodium methoxide in methanolat 70 °C, with a 12-h reflux. The residue was acidified topH 2.0, mixed with ddH2O and dissolved in 2.5 mol/L sulfuricacid in methanol for another 1 h reflux at 70 °C. The residuewas extracted twice with an equal volume of n-hexane, andthe hexane phase was dried over anhydrous Na2SO4 and an-alyzed by GC-MS. The GC program started at an oven tem-perature of 50 °C for 1 min, with subsequent increases of15 °C/min to 300 °C, and a final hold at 300 °C for 2 min.The nitrogen flow was 1 mL/min. The sample injection vol-ume was 1 μL. The PolarisQ GC/MS (Thermo FinniganThermo Scientific, Waltham, MA) was operated in full scanpositive ion mode covering the mass range of 70–400, with atotal scan time of 0.41 s. The MS scan start time was at6.6 min, and the ion source temperature was at 250 °C. Theinjector and detector (ion source) temperatures were 150 °Cand 230 °C, respectively. Heliumwas used as the carrier gas ata constant flow rate of 1.1 mL/min. Quantitation was per-formed by comparing integration data of M•+ fragment ionsresulting from the electron impact of the trimethylsilylesters of3-hydroxy fatty acids with those of the internal standard usingresponse factors previously determined using calibrationcurves made with pure standards. Experiments were conduct-ed in triplicate.

Ann Microbiol (2016) 66:1145–1156 1147

Surface tension assay

A automated surface/interfacial tensiometer (model ZL-2100;Shandong, China) was used to measure the surface tension ofthe supernatants of the F1 and F2 samples, respectively.Samples (20 mL) of different concentrations of F1 (1–900 mg/L) and F2 (1–90 mg/L) were placed onto the tensiom-eter platform. A platinum ring was slowly brought into contactwith the liquid–air interface in order to measure the surfacetension (mN/m). Between each measurement, the platinumring was rinsed three times with water and three times withacetone, and then allowed to dry. The effects of temperature,pH and NaCl concentration on the surface tension of RLswerealso measured for F1 and F2, respectively.

Emulsion test assay

The emulsion capacity of RLs was assessed as follows: 1 mgof RL (F1 or F2 RL species, respectively) was added to themixture of 5 mL atoleine and 5 mL water in a test tube,resulting in a RL concentration of 100 mg/L. After 30 s ofultrasonication, the tube was kept at 25 °C, and the sampleswere examined to determine emulsion ability (%) by the vol-ume fraction of emulsion layer in the total volume of samplesat different time-points. An equivalent amount (1 mg) of so-dium dodecyl sulfate (SDS) at 100 mg/L was also measuredwith the same procedure as control.

Optimization of nutrient and environmental conditionsfor RL production

Optimization studies were carried out for P. aeruginosaM408with regard to nutritional and environmental parameters. Thestarter culture was prepared by inoculating the strain in100 mL seed medium, followed by incubation on a shaker(180 rpm) at 28 °C for 24 h. The starter culture (2 %) wasinoculated into fermentation medium (pH 7.0) supplementedwith various carbon sources (olive oil, soybean oil, antoleine,glycerol, glucose and sucrose, respectively; all at 20 g/L) andvaried concentrations of olive oil (20–120 g/L) and nitrogensources (peptone, urea, ammonium sulfate, ammonium nitrateand sodium nitrate; all at 10 g/L). The cells were cultivated at28 °C with agitation (180 rpm) on a constant temperatureincubator shaker (model HZQ-F160; Beijing Donglian HarInstrument Manufacture Co., Ltd, Donglian, China) underaerobic conditions for 96 h. The surface tension of the culturesupernatants was measured using the Wilhelmy plate methodas described earlier. RLs were quantified by the anthrone sul-furic acid method (Smyth et al. 2010), and absorbance wasmeasured at 575 nm using rhamnose as standard after roughextraction of the RLs [an equal volume mixture ofchloroform:methanol (v/v, 2:1) was added to 50 mL fermen-tation broth after 10 min of centrifugation at 8000 rpm/min;

after 2 min of shaking, the lower chloroform phase was driedat 40 °C under vacuum; the yellow oil which remained is acrude extraction of the biosurfactants). After optimization ofmedium conditions, the effect of environmental factors wasstudied at different pH values (range pH 6–10), temperatures(range 23–43 °C) and agitation speeds (range 140–200 rpm).All RL production experiments were conducted in triplicateand the data presented as the mean ± standard error (SE).Characterization of the composition of the various RLs afteroptimization of cultivation were carried out as described inprevious sections.

Time-course study of RL production after optimization

After evaluating the nutritional and environmental parametersfor the production of RLs by P. aeruginosa M408, the time-course production of RLs was carried out under optimal con-ditions. The experiments were also conducted at a constanttemperature in an incubator shaker with a working volumeof 100 mL of the optimized medium and optimal conditions.Samples were collected at periodic intervals of 20 h, and esti-mation of RL production was carried out for a total fermenta-tion period of 180 h.

Results and discussion

Identification of biosurfactant-producing microorganism

We isolated three morphologically distinct microbial coloniesand ultimately identified isolate M408 as the highestbiosurfactant producer of these three strains. Further studies(oil displacement test to >5.0 mm in diameter and reduction inthe surface tension values to 27 mN/min) were conduted withstrain M408. The strain M408 was identified by 16S rRNAanalysis, and phylogenetic tree analysis suggested that thestrain belongs to the P. aeruginosa cluster (Fig. 1). The nucle-otide sequence of strainM408 has been deposited in GenBank(NCBI) under accession number HQ222816. This strainshares 99 % nucleotide similarity with P. aeruginosaKIZHAN1 and P. aeruginosa DSM 50071 T (accession num-bers KP244469 and LN681564, respectively). As such, weadded P. aeruginosa strain M408 to the list of biosurfactantproducers isolated from petroleum-contaminated sites.

Characterization of RLs

After preliminary isolation in the silica gel column to removeneutral lipids, the partially purified RL extracts from strainP. aeruginosa M408 were assayed and separated by TLC.Two different visualization bands, namely, F1 and F2, associ-ated to rhamnolipidic homologs with different mobility, were

1148 Ann Microbiol (2016) 66:1145–1156

observed (Fig. 2a). The Rf (retardation factor) values of thespots in bands F1 and F2 were 0.12 and 0.45, respectively.

The chemicals in the corresponding band, recovered by scrap-ing and eluted from the silica gel with methanol and chloroform,were identified by ESI–MS/MS. The glycolipid chemicals werecomposed of L-rhamnose and β-hydroxydecanoic acid and β-hydroxy dodecanoic acid (Fig. 2b).

The ESI–MS/MS analyses resulted in the identificationof three rhamnolipidic compounds, corresponding to in-tense signals of pseudo-molecular ions at m/z 650 and678 in band 1 (F1), and 504 in band 2 (F2), respectively(Fig. 3). Among these, the pseudo-molecular ion at m/z 1299appeared to be a dimeric agglomerate of m/z 650 formed duringthe electrospraying process. These ions are in agreement withthe structures expected for L-rhamnopyranosyl–L-rhamnopyranosyl–β-hydroxydecanoyl–β-hydroxydecanoate( R h a - R h a - C 1 0 -C 1 0 ) , L - r h am n o p y r a n o s y l – L -r h a m n o p y r a n o s y l – β - h y d r o x y d e c a n o y l – β -hydroxydodecanoate (Rha-Rha-C10-C12/Rha-Rha-C12-C10)and L- rhamnopyranosy l–β -hydroxydecanoyl–β -hydroxydecanoate (Rha-C10-C10). Analyses of the main massfragments verified the expected molecular formula of therhamnolipidic compound detected in the TLC band, of whichthe main pseudo-molecular ion and ion fragments were deter-mined (Table 1).

These results revealed that the RL mixture produced bythe isolated strain P. aeruginosa M408 contained only fourtypes of RLs (Rha-Rha-C10-C10, Rha-Rha-C10-C12,Rha-Rha-C12-C10 and Rha-C10-C10) when olive oil wasthe sole carbon source. This result differed from those ob-tained with previously reported P. aeruginosa strains forwhich characterization of the RL products was less specific(George and Jayachandran 2009; Xia et al. 2012). In addition,a number of published studies have reported thatP. aeruginosa cultured with carbon sources other than olive

oil often produced 5–20 different structures of RL compo-nents (Table 2). In general, the physical and chemical proper-ties of a surfactant product is determined by its RL

Pseudomonas aeruginosa M408 (HQ222816)

Pseudomonas aeruginosa (KP244469)

Pseudomonasa eruginosa (LN681564)

Pseudomonas aeruginosa (KJ854401)

Pseudomonas resinovorans (AB088750.2)

Pseudomonas otitidis (HQ851079)

Pseudomonas otitidis (KC540914)

Pseudomonas azelaica (AM088475)

Pseudomonas pseudoalcaligenes (AY880303)

Pseudomonas stutzeri (PSU26416)

Pseudomonas stutzeri (AF054935)10080

100

98

75

100

75

0.005

Fig. 1 Phylogenetic tree predicted using 16S rRNA gene sequences bythe neighbor joining method. The bootstrap included 1000 replicates.Strain HQ222816 belongs to the Pseudomonas aeruginosa cluster. Taxa

are represented by type strains with their respective GenBank accessionnumber. Scale bar Expected number of substitutions averaged over all ofthe analyzed sites, number in parenthesis Accession number

O

HO

O

I

O

OHO

II

a b

F2

F1

Fig. 2 Characterization of the rhamnolipids (RLs) by thin-layerchromatography (TLC) and gas chromatography–mass spectrometry(GC-MS). a TLC analysis of RLs produced by P. aeruginosa M408with visualization by 2,7-dichlorofluorescein. F2 First homologoussamples of RL species eluted from the column using the mobile phasesCHCl3:CH3OH (180:25, v/v), F1 other samples of RL speciessubsequently eluted from column using pure methanol as the mobilephase. b GC-MS spectra of fatty acid components of the RL producedby P. aeruginosa M408. I β-Hydroxydecanoic acid methyl ester, II β-Hydroxydodecanoic acid methyl ester

Ann Microbiol (2016) 66:1145–1156 1149

composition. Therefore, the direct production of more specificRL products would improve downstream, high-cost purifica-tion procedures. The selective synthesis of differentrhamnolipids may also be useful for studies on the functionalproperties of single components of commercial derivatives.As such, P. aeruginosa M408 may be an ideal candidate forthe production of RLs of simple composition for industrialapplications.

Surface activity of RLs produced by P. aeruginosa M408

The surface activity of RLs obtained from the supernatant ofbatch cultures of P. aeruginosa M408 was characterized bymeasuring the surface tension. As illustrated in Fig. 4, surface

////

OOH

OH OH

CH3

OOH

OH O

CH3

O CH

(CH2)6

CH2

CH3

C

O

O CH

O

OHCH2

////

(CH2)6

CH3

OOH

OH OH

CH3

OOH

OH O

CH3

O CH

(CH2)6

CH2

CH3

C

O

O CH C

O

OHCH2

(CH2)8

CH3 OOH

OH OH

CH3

OOH

OH O

CH3

O CH

(CH2)8

CH2

CH3

C

O

O CH C

O

OHCH2

(CH2)6

CH3

OOH

OH OH

CH3

O CH

(CH2)6

CH2

CH3

C

O

O CH C

O

OHCH2

(CH2)6

CH3

a

b

163 479 169

163479 197

163507 169

163333 169

Fig. 3 Mass spectrometryspectrograms of rhamnolipidspurified from batch cultures ofP. aeruginosaM408. a F1 L-rhamnopyranosyl–L-rhamnopyranosyl–β-hydroxydecanoyl–β-hydroxydecanoate (top; Rha-Rha-C10-C10), L-rhamnopyranosyl–L-rhamnopyranosyl–β-hydroxydecanoyl–β-hydroxydodecanoate (bottom;Rha-Rha-C10-C12/Rha-Rha-C12-C10), b F2L-rhamnopyranosyl–β-hydroxydecanoyl–β-hydroxydecanoate (Rha-C10-C10)

Table 1 Composition of the mixture of rhamnolipids produced byPseudomonas aeruginosa M408 using olive oil as the sole carbonresource

Samplea Structureb Key fragments (m/z) [M-H]−

F1 Rha-Rha-C10-C10 163, 169, 479 650

Rha-Rha-C10-C12 163, 197, 479 678

Rha-Rha-C12-C10 163, 169, 507 678

F2 Rha-C10-C10 163, 169, 333 504

Rha L-rhamnopyranosyl, RLs rhamnolipidsa For explanation of F1 and F2 samples, see Isolation, purification andcharacterization of RLs section and caption to Fig. 2b For more detail on structure, see Characterization of RLs section andFig. 3

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tension rapidly decreased from 72mN/m (for pure water) withincreasing concentration of RLs, reaching aminimum value ofapproximately 31 mN/m for F1 samples and 27 mN/m for F2samples. A critical micelle concentration (CMC) of approxi-mately 120 mg/L for F1 samples and 60 mg/L for F2 sampleswas estimated from Fig. 4. The CMC values thus estimateddiffer from those reported earlier for RLs (27–54 mg/L), prob-ably due to the specific component of RL products (Bodourand Miller-Maier 1998).

Emulsion capacity of RLs produced by P. aeruginosaM408

Figure 5 indicates the emulsion capacity of RLs for atoleineusing SDS as the control, showing that a small amount of RLsresulted in a significant emulsification of the atoleine.Approximately 100 mg/L of RLs was sufficient to reach anemulsion capacity of 25 and 42 % for atoleine and watermixture, respectively, even after 96 h. Figure 5 also showsthat after about 96 h of incubation, F2 RLs had a better

emulsification ability than F1 RLs. One explanation for thisdifference may be the higher polarity of RLs in the F1 samplesdue to the higher content of rhamnose components in the RLsof the F1 samples compared to the F2 samples. Emulsioncapacities of 87 and 90 % with atoleine for the F1 and F2samples, respectively, after 24 h at a low concentration of100 mg/L (Fig. 5) together with the surface activity results(Fig. 4) clearly demonstrate the potential of using thebiosurfactant in a variety of commercial applications, suchas bioremediation of oil pollutants (Mulligan 2005).

Effect of nutrients and environmental parameters on RLproduction by P. aeruginosa M408

Effect of carbon sources on RL production by P. aeruginosaM408

The type of carbon substrate has been reported to markedlyaffect the production yield of RLs (Lang and Wullbrandt1999; Maier and Soberon-Chavez 2000). Consequently, we

Table 2 Composition of rhamnolipid mixture produced by P. aeruginosa strains when cultured with different carbon sources

Carbon sources Components of RLs Reference

Mono-RLs Di-RLs

Olive oil Rha-C10-C10 Rha-Rha-C10-C10Rha-Rha-C10-C12Rha-Rha-C12-C10

Present study

Glycerol Rha-C8-C10Rha-C10-C8Rha-C10-C10Rha-C10-C12Rha-C12-C10Rha-C10-C12:1

Rha-Rha-C8-C10Rha-Rha-C10-C8Rha-Rha-C10-C10Rha-Rha-C10-C12Rha-Rha-C12-C10Rha-Rha-C10-C12:1

Monteiro et al. (2007)

Corn oil Rha-C10-C10 Rha-Rha-C10-C8Rha-Rha-C10-C10Rha-Rha-C10-C12Rha-Rha-C10-C12:1

Mata-Sandoval et al. (1999)

Glucose Rha-C8-C10Rha-C10-C8Rha-C10-C10Rha-C12-C10Rha-C10-C12Rha-C10-C12:1Rha-C12-C12

Rha-Rha-C8-C10Rha-Rha-C10-C8Rha-Rha-C10-C10Rha-Rha-C10-C12RhaRha-C12-C10Rha-Rha-C10-C12:1Rha-Rha-C12-C12

Rendell et al. (1990)

Mannitol Rha-C8-C8Rha-C10-C10

Rha-Rha-C8-C8Rha-Rha-C10-C10Rha-Rha-C12-C12

Nayak et al. (2009)

Palm oil Rha-C8-C10Rha-C10-C8Rha-C10-C10Rha-C10-C12Rha-C12-C10Rha-C10-C12:1Rha-C12:1-C10

Rha-Rha-C8-C10Rha-Rha-C10-C8Rha-Rha-C12:1-C12Rha-Rha-C10-C14:1

Pornsunthorntawee et al. (2008)

Soapstock Rha-C10-C10Rha-C10-C12Rha-C10-C12:1

Rha-Rha-C10-C10Rha-Rha-C10-C12Rha-Rha-C10-C12:1

Benincasa et al. (2004)

Ann Microbiol (2016) 66:1145–1156 1151

tested the effectiveness of six carbon sources, including oliveoil, soybean oil, atoleine, glycerol, glucose and sucrose, on RLproduction by P. aeruginosaM408. The results show that thehighest yields of RLs, namely, 1.84 and 0.70 g/L, were ob-tained with olive oil and soybean oil, respectively, as the solecarbon source, after 96 h of cultivation. Cultivation ofP. aeruginosa M408 with the other carbon sources (e.g.atoleine, glycerol, glucose and sucrose) resulted in a RL pro-duction of <0.60 g/L at the end of the fermentation period(Fig. 6a). These results suggest that carbon sources such asolive oil and soybean oil may be effective substrates for theproduction of RLs. Maier and Soberon-Chavez (2000) foundvegetable oils, such as corn oil, olive oil, and soybean oil, tobe very effective in RL production, as did Pimienta et al.(1997) who noted that a very high yield of RLs (1.4 g/L)was obtained when vegetable oil was used as the carbon

substrate. In our study, the most effective carbon source forRL production was olive oil.

Effect of nitrogen source and the C/N ratio on RL productionby P. aeruginosa M408

The effect of various nitrogen sources (all at 10 g/L;(NH4)2SO4, NH4NO3, NaNO3, peptone, urea) on RL produc-tion by P. aeruginosa M408 cultivated in fermentation medi-um supplemented with 20 g/L olive oil was tested; the resultsshown in Fig. 6b. As shown, compared to the RL yield infermentation medium with only 20 g/L olive oil, the yield ofRLs increased by approximately threefold, to 6.65 g/L, withthe addition of sodium nitrate; in comparison, the addition ofurea resulted in a very low RLyield (0.35 g/L). RL productionfollowing the addition of peptone was similar to that withsodium nitrate, but the high cost of peptone restricts itslarge-scale use, indicating that sodium nitrate was the appro-priate nitrogen source for further studies. The influence ofdifferent nitrogen sources on the production of RLs has beenwell established in previous studies, with results suggestingthat the presence of NO3

− promotes the production of RLswhereas that of NH4

+ inhibits RL production (Mulligan andGibbs 1989; Ramana and Karanth 1989). Several studies havedemonstrated that NO3

− effectively elicits more rhlAB expres-sion than NH4

+, leading to the suggesting that NO3− can be

considered to be a suitable and good nitrogen source for RLproduction (Arino et al. 1996). On the other hand, high levelsof NH4

+ have been found to reduce the formation of RLs, andthis can be correlated with lower glutamine synthase activity(Totten et al. 1990). Based on our identification of sodiumnitrate as a suitable nitrogen source for RL production, westudied the effect of the mass ratio of carbon and nitrogensources on the production of RLs (Fig. 6c). In this study, wevaried the sodium nitrate concentration between 2 and 10 g/Lin combination with 20 g/L olive oil, keeping all other

Time (h)

Em

ulsi

fyin

g ca

paci

ty (%

)

Fig. 5 Emulsifying capacity of RLs in the F1 samples, RLs in the F2samples and sodium dodecyl sulfate (SDS) as control at a concentration of100 mg/L

0 200 400 600 800 1000

30

40

50

60

70

80

Rhamnolipid (mg/L)0 20 40 60 80 100

20

30

40

50

60

70

80

Sur

face

tent

ion

(mN

/m)

Sur

face

tent

ion

(mN

/m)

Rhamnolipid (mg/L)

a b

Fig. 4 Surface tensions of F1 and F2 samples in solution at different concentrations. a RLs in F1 samples, b RLs in F2 samples

1152 Ann Microbiol (2016) 66:1145–1156

components constant. A maximum RL production of 11.25 g/L (with 20 g/L olive oil added) was recorded with an optimalC/N ratio of 8. These results are similar to those reportedearlier with P. aeruginosa 44 T1 (Manresa et al. 1991) andP. aeruginosa 47 T2 NCIB 40044 (Haba et al. 2000).

Additionally, with a fixed C/N ration at 8, the effect of theolive oil concentration on RLs production was investigated(Fig. 6a inset). It shows a general trend that RLs productioninitially increased with increasing carbon substrate concentra-tion, until it reached a maximum value and then leveled off.The maximum RLs yield was obtained at 12.44 g/L when theconcentration of olive oil was 40 g/L.

Effect of environmental parameters on RL productionby P. aeruginosa M408

The impact of the pH of the initial medium and incubationtemperature on yield of RLs was investigated. The growth andproduction of RLs by P. aeruginosaM408 in medium at var-ious initial pH values are shown in Fig. 7a. The results indicatethat maximum yield of RLs (with 20 g olive oil added per liter

medium) was achieved at pH 7.0. Most Pseudomonasaeruginosa strains produce RLs best at an incubation temper-ature of between 27 °C and 37 °C (Wei et al. 2005; Kumaret al. 2012; Nalini and Parthasarathi 2014). The effect of tem-perature on the growth and yield of RLs is shown in Fig. 7b.Maximum production of RLs was observed at 28 °C, with ayield of 12.10 g in the presence of 20 g olive oil/L medium.Agitation rate affects the level of dissolved oxygen and masstransfer in the fermentation medium, with generated RLs in-creasing the dissolved oxygen levels, and both factors alsohave a significant impact on the bacteria. Such factors arecrucial to the cell growth and biosurfactant formation of thestrictly aerobic bacterium P. aeruginosa M408, especiallywhen cultivated in a shake flask. For oil substrates, raisingthe rotation speed not only increases the oxygen mass transferrate in the fermentation liquid, but also increases the frequencyof contact with oil substrates and bacterial cells. RL produc-tion at various agitation speeds are shown in Fig. 7c, withmaximum production of RLs occurring at 200 rpm, with ayield of 12.32 g in the presence of 20 g olive oil/L medium.These results also reveal that the production of RLs was

olive oil soybean oil atoleine glycerol glucose sucrose0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

Rha

mno

lipd

(g/L

)

Carbon sources (20 g/L)(NH4)2SO4 NH4NO3 NaNO3 peptone urea

0

1

2

3

4

5

6

7

Nitrogen sources (10 g/L)

Rha

mno

lipd

(g/L

)

2 4 6 8 100

2

4

6

8

10

12

C source/N sources (mass ratio)

Rha

mno

lipd

(g/L

)

a b

c

20 40 60 80 100 1200

2

4

6

8

10

12

14

Olive oil (g/l)

Rha

mno

lipd

(g/l)

Fig. 6 Effect of different carbon (C) sources (a), different nitrogen (N) sources (b) and the C/N ratio (c) on the production of RLs by P. aeruginosaM408. a Inset Effect of olive oil concentrations on RL yield. Note that different scales for RL production ( y-axis) are used in the different figure parts

Ann Microbiol (2016) 66:1145–1156 1153

gradually enhancedwith an increase in agitation speed but thatabove 180 rpm, higher agitation speeds did not clearly im-prove RL production. We therefore used 200 rpm as our opti-mal environmental condition.

After the optimization of nutritional and environmentalconditions, the production of RLs increased by approximatelysevenfold. RLyield was 1.84 g/20 g of bioglycerol used undernon-optimized conditions (fermentation medium supplement-ed with 2% olive oil at pH 7.0, 28 °C and 180 rpm) and 12.6 gunder the optimized conditions (40 g olive oil/L medium andoptimal conditions determined in this study); the latter value isa 6.85-fold increase over the non-optimized conditions. Theoptimal medium composition consisted of 1.0 g/L MgSO4 ·7H2O, 1.1 g/L KCl, 3.4 g/L KH2PO4, 4.4 g/L K2HPO4 ·3H2O, 1.1 g/L NaCl, 5.0 g/L NaNO3, and 40 g/L olive oil;the optimized environmental conditions were pH 7.0, a tem-perature of 28 °C and an agitation speed of 200 rpm for 96 h.The RL production level reported here under the optimizedconditions is higher than previously reported values (1.4–10.0 g/L) obtained from using olive oil as the carbon substrate(Lang and Wullbrandt 1999; Wei et al. 2005), suggesting thatP. aeruginosaM408 is a high RL producing strain. However,

metabolically engineered P. aeruginosa PAO1 usingvitreoscilla hemoglobin technology has achieved a maximumyield of 39 g/L with 250 g/L sunflower oil as carbon source(Dobler et al. 2016).

Time-course study of RL production by P. aeruginosaM408

The kinetics profile of P. aeruginosa M408 with reference torhamnolipid production during culture growth in the opti-mized medium is shown in Fig. 7d. A maximum RL yield of12.6 g/L was observed at 96 h of culture, with sharp increasebeginning at 24 h of culture. After 96 h of culture, a slightdecrease in RL content was observed, following which theproduction level remained almost constant. The curve profileis similar to that reported for P. aeruginosa J4, but with aslightly different timeline to achieve maximumRL production(Wei et al. 2005). These similar findings for bothP. aeruginosaM408 and P. aeruginosa J4 might verify previ-ous findings that RL is a secondary metabolite secreted byP. aeruginosa in the stationary phase of growth (Maier andSoberon-Chavez 2000).

Rha

mno

lipd

(g/L

)

6 7 8 9 100

2

4

6

8

10

12

14

pH23 28 33 38 43

Temperature (°C)

0

2

4

6

8

10

12

14

Rha

mno

lipd

(g/L

)

140 160 180 200

Agitation speed (rpm)

0

2

4

6

8

10

12

14

Rha

mno

lipd

(g/L

)

0 20 40 60 80 100 120 140 160 180

Incubation time (h)

0

2

4

6

8

10

12

14

Rha

mno

lipd

(g/L

)

a b

c d

Fig. 7 Effect of environmental parameters on RL yield of P. aeruginosa M408 grown in fermentation medium with olive oil as the carbon source. aInitial pH values, b incubation temperature, c agitation speed, d incubation time

1154 Ann Microbiol (2016) 66:1145–1156

Conclusions

We report the successful isolation of a bacterial strain(P. aeruginosaM408) capable of producing RLs from variouscarbon sources. Pseudomonas aeruginosa M408 was foundto be capable of degrading vegetable oils as well as mineraloils (e.g. diesel and kerosene) to produce biosurfactants.Hence, the strain itself or its biosurfactant products are poten-tial candidates for use in the bioremediation of oil pollutants.Among the seven substrates of various carbon sources exam-ined, olive oil was the most efficient one for RL production.At a concentration of 40 g olive oil/L medium, a maximumRL production level of 12.6 g/L was reached. RL productionwas optimized in batch cultures, when the temperature andagitation rate were controlled at 28 °C and 200 rpm, respec-tively. RLs in the culture broth were purified primarily viasolvent extraction. The ESI-MS/MS analysis showed thatthe purified product contained three types of RLs compounds.RLs produced by P. aeruginosa M408 had a critical micelleconcentration of 120 mg/L for F1 samples and 60 mg/L for F2samples and exhibited an excellent emulsification capacity foratoleine (87 and 90 % for F1 and F2 samples, respectively,after 24 h).

Acknowledgments Wewould like to thank the DUT fundamental fundfor supporting this work. (Fund Number: DUT13RC(3)072)

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