Biosurfactant production by Pseudomonas aeruginosa SP4 using sequencing batch

Biochemical Engineering Journal 49 (2010) 185–191

Contents lists available at ScienceDirect

Biochemical Engineering

Journal

j o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /b e

Biosurfactant production by Pseudomonas aeruginosa SP4using sequencing batch reactors:Effect of oil-to-glucose ratio

Sira Pansiripat a ,Orathai Pornsunthorntawee a ,Ratana Rujiravanit a ,b ,Boonyarach Kitiyanan a ,b ,Pastra Somboonthanate a ,b ,Sumaeth Chavadej a ,b ,?

a The Petroleum and Petrochemical College,Chulalongkorn University,Bangkok 10330,Thailand

Center for Petroleum,Petrochemicals,and Advanced Materials,Chulalongkorn University,Bangkok 10330,Thailand

a r t i c l e i n f o Article history:

Received 27October 2009Received in revised form 17December 2009

Accepted 18December 2009

Keywords:

Biosurfactants

Pseudomonas aeruginosa

Sequencing batch reactor (SBR)Glycolipids Rhamnolipids

a b s t r a c t

Sequencing batch reactors were used for biosurfactant production from Pseudomonas aeruginosa SP4.The lab-scale aerobic sequencing batch reactor units were operated at an oil loading rate of 2kg/m 3day,a cycle time of 2days/cycle,and a temperature of 37?C.A mineral medium with palm oil was used as the culture medium.Glucose,a supplemental carbon source,was added for enhancing the microbial growth,which,in turn,gave a better process stability.The optimum oil-to-glucose ratio for the biosurfactant production was 40:1,providing a surface tension reduction of 58.5%,a chemical oxygen demand removal of 85.1%,and an oil removal of 77.7%.The maximum biosurfactant concentration in the bioreactors was about 1.1times the critical micelle concentration.The biosurfactant product was predominantly composed of Rha-Rha-C 8-C 10and Rha-Rha-C 10-C 8,and its critical micelle concentration was 150mg/l.

1.Introduction

An ef?cient and cost-effective bioprocess for biosurfactant pro-duction is necessary for expanding the commercial utilization of biosurfactants by increasing the production yield and reducing the cost of biosynthesis and downstream processing [1,2].Sequencing batch reactors (SBRs),which are ?ll-and-draw systems widely used for the treatment of both domestic and industrial wastewaters,are of great interest for adaptation for biosurfactant production from appropriate substrates on a large productivity https://www.360docs.net/doc/7215077129.html,pared to continuous-?ow stirred tank reactors (CSTRs),SBRs show better reactor performance and give a greater biosurfactant production [3].Cassidy and Hudak [4]found that an SBR encourages micro-bial growth,thus resulting in the enhancement of the biosurfactant production to nearly 70times the critical micelle concentration (×CMC).In addition,it was recently reported that biosurfactant production in an SBR system can be operated to reach a maximum at the end of the operating cycle [5].

For the production of microbial metabolites on a large scale,it is important to know the regulation mechanisms of the cho-

?Corresponding author at:The Petroleum and Petrochemical College,Chula-longkorn University,Soi Chulalongkorn 12,Phayathai Road,Pathumwan,Bangkok 10330,Thailand.Tel.:+6622184139;fax:+6622184139.

E-mail address:sumaeth.c@chula.ac.th (S.Chavadej).sen microorganism in order to achieve a high production yield [6].For certain species of Pseudomonas ,the carbon source plays a key role in the biosurfactant production.The Pseudomonas species are able to utilize both water-soluble carbon sources (such as glyc-erol,glucose,mannitol,and ethanol)[2,7–9]and water-immiscible substrates (like n -alkane and vegetable oils)[2,10–12]for the biosurfactant production.However,the carbon source preference also depends on the chosen bacterial strain.For example,Mata-Sandoval et al.[13]reported that a greater biosurfactant yield was obtained when hydrophobic carbon sources (long chain alcohol or corn oil)were used in the biosurfactant production by Pseudomonas aeruginosa UG2.In contrast,Wu et al.[14]found that hydrophilic substrates (glucose and glycerol)were superior in terms of both yield and productivity in the biosurfactant production by P.aerug-inosa EM1.Interestingly,when both hydrophilic and hydrophobic substrates were used simultaneously,the biosurfactant produc-tion from the Pseudomonas species was found to be signi?cantly increased [15,16].

In this present study,the SBR process was used for biosurfactant production by P.aeruginosa SP4isolated from petroleum-contaminated soil in Thailand [17].The biosurfactant production was carried out in two identical lab-scale aerobic SBR units.A min-eral medium (MM)and palm oil were used as the nutrient and as the hydrophobic carbon sources,respectively.The aim of the present work was to promote the microbial growth and to improve the process performance of the studied SBR units;therefore,glucose,a

186S.Pansiripat et al./Biochemical Engineering Journal49 (2010) 185–191

supplemental hydrophilic carbon source,was added to the culture medium.The effect of oil-to-glucose ratio on the process perfor-mance was investigated,and the biosurfactant concentration in the SBRs operated under the optimum oil-to-glucose ratio was subse-quently determined by using the critical micelle dilution(CMD) method.The biosurfactant was extracted from the ef?uent sam-ple,and the predominant components in the biosurfactant product were characterized.

2.Materials and methods

2.1.Materials

The nutrient broth(Difco,USA)was prepared at a?xed concen-tration of8g/l,as recommended by the supplier.The palm oil was purchased from Morakot Industry,Co.,Ltd.(Thailand).Acetonitrile (HPLC grade)was supplied by Labscan Asia Co.,Ltd.(Thailand).Tri-?uoroacetic acid,TFA,(≥98.0%purity)was purchased from Fluka (USA),and d-glucose anhydrous was supplied by Ajax Finechem (Australia).The MM was modi?ed from that of Dubey and Juwarkar [18].It contained NaNO3(0.5g/l),KH2PO4(0.5g/l),K2HPO4(0.5g/l), MgSO4·7H2O(0.5g/l),KCl(0.1g/l),and FeSO4·7H2O(0.01g/l).The C:N and C:P ratios in the feed solutions were kept constant at16:1 and14:1,respectively,because these two ratios were previously reported as the optimum ratios for maximum biosurfactant pro-duction by P.aeruginosa DSM2659[7].All chemicals were used as received,without further puri?cation.

2.2.Bacterial strain and culture growth conditions

P.aeruginosa SP4was isolated from petroleum-contaminated soil in Thailand[17].The isolated strain was maintained on nutri-ent agar slants at4?C to minimize the biological activity,and was subcultured every month.

2.3.Con?guration of the sequencing batch reactors

The SBR units used in this present study consisted of a round-bottom vessel with a total volume of3000ml and a working volume of1500ml.The SBRs were operated under aseptic conditions,and the temperature was controlled at37±1?C by circulating warm water through the water jacket of the bioreactor.There were two feeding lines,one of palm oil and the other of MM(either with or without glucose),using a peristaltic pump and a diaphragm pump, respectively.The500ml feed solution to each bioreactor was con-trolled by using a level controller with a timer.A solenoid valve with a timer was used to control the discharge volume(500ml)?owing to a product https://www.360docs.net/doc/7215077129.html,pressed air,which was?ltered through a 0.2-?m pore size?lter before entering the bioreactor,was used to achieve the aerobic condition and the complete mixing inside the bioreactor.Three timers,one connected to the feed pumps,one to the air pump,and one to the solenoid valve,were used to set feeding,reaction or aeration,and decanting times,respectively.

2.4.Reactor start-up and operating conditions

For the reactor start-up,an inoculum was prepared by transfer-ring the preserved bacterial colonies into a series of three250-ml Erlenmeyer?asks,each containing50ml of the nutrient broth.The culture was incubated at37?C in a shaking incubator at200rpm for22h[17].Then,150ml of the as-prepared inoculum was trans-ferred to each SBR unit.The two SBRs were fed the palm oil and the MM(either with or without glucose)until a working volume of 1500ml each was reached.The operation was set constant at an oil loading rate(OLR)of2kg/m3day and a cycle time of2days/cycle—a feeding step of5min,a reaction step of47h,a sedimentation step of50min,and a decanting step of5min[5].The effect of the oil-to-glucose ratio on the process performance was studied at60:1, 40:1,30:1,20:1,and10:1,while the SBR operation in the absence of the glucose addition was used as a control.The draining volume of each SBR unit was kept constant at500ml.Therefore,500ml of the mixture of the MM(either with or without glucose)and the palm oil used as the feed was introduced to the reactor during the feed-ing step,and the liquid holding volume was brought from1000to 1500ml,which was the working volume of each reactor.Aeration was provided throughout the reaction step by using compressed air, and was shut off during the sedimentation and the decanting steps to allow the bacterial cells to settle.Only500ml of the clari?ed supernatant was discharged from the reactor during the decant-ing step.All of the ef?uent samples were taken for analysis during a steady-state operation,which could be justi?ed as the ef?uent chemical oxygen demand(COD)remained constant.The optimum oil-to-glucose ratio was then selected on the basis of surface ten-sion reduction,COD removal,oil removal,mixed liquor suspended solids(MLSS),and ef?uent suspended solids(SS).

2.5.Extraction of the biosurfactant product

An ef?uent sample taken from the bioreactors operated under the optimum oil-to-glucose ratio was centrifuged at4?C and 8500rpm for20min in order to remove the microbial cells.The obtained supernatant was treated by acidi?cation to pH2.0using a 6M HCl solution,and the acidi?ed supernatant was left overnight at 4?C for the complete precipitation of the biosurfactant[19].After centrifugation,the precipitate was dissolved in a0.1M NaHCO3 solution,followed by the biosurfactant extraction step with a solvent having a2:1CH3Cl–C2H5OH ratio at room temperature (25–27?C)[20].The organic phase was transferred to a round-bottom?ask connected to a rotary evaporator in order to remove the solvent at40?C,yielding a viscous honey-colored biosurfactant product.About720mg of the biosurfactant was extracted per liter of ef?uent.

2.6.Analytical methods and measurements

2.6.1.Surface tension measurement

A sample of either the ef?uent or the mixed liquor taken from the bioreactors was centrifuged at4?C and8500rpm for20min in order to remove the microbial cells.The surface tension of the obtained supernatant was measured by using a drop shape analysis system(Krüss,DSA10Mk2)and was carried out at room temper-ature using the pendant drop method.(For the calibration of the instrument,the surface tension of pure water was?rst measured.) The measurement was repeated at least3times,and an average was used to express the surface tension of the sample.A higher bio-surfactant concentration in a test sample provides a lower surface tension until CMC is reached.To study the biosurfactant produc-tion in the SBR units,the percentage reduction of surface tension is calculated by the following equation:

%surface tension reduction=

( m? c)

×100

where m is the surface tension of the feed solution and c is the surface tension of the centrifuged sample.

The surface tension measurement was also employed to quantify the biosurfactant concentration in the reactor using the CMD technique[21].When the surfactant concentration is greater than the CMC,the surface tension remains constant at some minimum value.The surface tension increases only when the surfactant concentration drops below the CMC upon dilu-tion.In this present study,the minimum surface tension was in the range of28–30mN/m.Thus,surface tension was mea-

S.Pansiripat et al./Biochemical Engineering Journal49 (2010) 185–191187 sured at different dilution ratios until the surface tension rose

above30mN/m.This dilution ratio was subsequently used to

calculate the biosurfactant concentration,which is expressed as

×CMC.

To study the surface activities of the biosurfactant extracted

from the ef?uent sample in terms of surface tension reduction

and CMC,the surface tension of the aqueous solution at different

biosurfactant concentrations was measured.Then the CMC of the

extracted biosurfactant was determined from the break point of the

surface tension versus its log of bulk concentration curve.

2.6.2.Quanti?cation of the glucose consumption

To determine the amount of glucose consumed by the bacterial

culture,the glucose concentration in the ef?uent was quanti-

?ed by using a Glucose(HK)Assay Kit(GAHK-20,Sigma–Aldrich

(USA)).Sample preparation followed the technical bulletin from

the supplier.Brie?y,an ef?uent sample was centrifuged at4?C

and8500rpm for20min to remove the microbial cells,and the

obtained supernatant was diluted10times with deionized water.

Then,100?l of the diluted sample was added to1ml of a glucose

assay reagent in a test tube.After thoroughly mixing the sample,

the tube was incubated at room temperature for15min.After that,

its absorbance(A test)was measured at a wavelength of340nm by

using a UV–vis spectrophotometer(Shimadzu,UV-2550).The dif-

ference between the absorbance of the sample(A sample blank)and

the glucose assay reagent(A reagent blank)can be used to indicate the

glucose concentration in a sample.The sample blank was prepared

by adding100?l of the diluted sample to1ml of deionized water in

a test tube,while the reagent blank was prepared by adding100?l

of deionized water to1ml of the glucose assay reagent.The glucose

concentration in the test sample is calculated in terms of mg/ml of

glucose by the following equation:

mg/ml of glucose=(0.029)( A)(TV)(F)

(SV)

where A=A test?(A sample blank+A reagent blank);TV:total assay vol-ume(ml);SV:sample volume(ml);and F:dilution factor from sample preparation.

2.6.

3.Quanti?cation of the palm oil

The palm oil concentrations in both the feed and the ef?u-ent samples were determined by the partition-gravimetric method using dichloromethane as a solvent[22].Brie?y,samples were acid-i?ed with1:1HCl to pH2and then centrifuged at12,000rpm for10min.The supernatant was then extracted with30ml of dichloromethane in a separator funnel.After shaking for2min,the separator funnel was left standing to obtain two separate phases. The lower layer(solvent phase)was drained through another fun-nel with a?lter paper(Whatman No.40)with10g of Na2SO4,and both the separator funnel and the?lter paper were rinsed with the solvent.The upper layer(aqueous phase)was re-extracted a few more times using the same procedure.The dichloromethane was evaporated from the?ltered extracted solvent phase at ambi-ent temperature overnight.The resultant residue was weighed to represent the palm oil content in the test sample,and the sub-strate consumption by the microbe was calculated to express the oil removal percentage.

2.6.4.Measurement of microbial concentration

The MLSS and the SS were used to represent the microbial cell concentration in the bioreactors and the cell wash-out,respec-tively.The analysis of the MLSS and the SS followed standard methods[22].Brie?y,the samples taken from the bioreactor during either the reaction or the decanting step were?ltered through a0.2-?m pore size?lter and washed with distilled water.The residues retained on the?lter were dried at105?C for at least24h to obtain

a constant weight.

2.6.5.Chemical analyses

The COD is a parameter indicating the concentration of organic compounds in a sample.It is expressed in terms of the amount of oxygen required for the chemical oxidation using K2Cr2O7.The COD measurement was done by using a COD reactor(HACH,45600) and a spectrophotometer(HACH,Dr/2700).In addition to the COD measurement,total organic carbon(TOC)analysis was used to determine both the C:N ratio and the C:P ratio in the feed solutions. The TOC was done by using a TOC analyzer(Shimadzu,TOC-Vcsh). Both the total nitrogen(TN)and the total phosphorus(TP)were measured using a spectrophotometer(HACH,Dr/2700).

2.6.6.Analysis of the biosurfactant components

The components in the biosurfactant extracted from the ef?u-ent samples were analyzed by using a high performance liquid chromatography(HPLC)instrument(an Alltech580autosampler; an Alltech HPLC pump,model626;and an Inertsil?ODS-3col-umn)equipped with an evaporative light scattering detector(ELSD) (Alltech,2000ES).The mobile phase solutions were an aqueous solution of10%acetonitrile(A)and pure acetonitrile(B).Both elu-ents contained0.1%TFA.The gradient system was used,starting with B from30%to70%in5min and then from70%to90%in 15min.After that,the gradient of B was raised again to100%at the end of the process[23].The?ow rate of the mobile phase was set constant at0.5ml/min,and the sample injection volume was 50?l.The ELSD drift tube temperature was maintained at100?C, while the nebulizer?ow rate was kept constant at1.5l/min[24]. The gradual change in the af?nity of the mobile phase resulted in the fractionation of the biosurfactant components.

2.6.7.Statistical analysis

All of the experimental data,which were taken under steady-state conditions,are expressed in terms of arithmetic averages obtained from at least three replicates,and the analyses were done using SigmaPlot software,version8.02(SPSS Inc.,UK).

3.Results and discussion

3.1.Effect of oil-to-glucose ratio

In the present work,the in?uence of oil-to-glucose ratio on the biosurfactant production by P.aeruginosa SP4using the SBR sys-tem was investigated.The surface tension reduction during the SBR operation was used to indicate the biosurfactant production in the studied SBR units,while the COD removal represents the utiliza-tion of carbon source–palm oil,either with or without glucose–by the bacterial culture.The oil removal is a direct measurement parameter of the palm oil consumption.From the results of the glucose quanti?cation,it was found that the strain SP4completely consumed the added glucose at all?ve investigated oil-to-glucose ratios.Table1lists the characteristics of the feed solutions at the ?ve different oil-to-glucose ratios,compared to those without glu-cose addition.The in?uent COD mainly came from the added palm oil and glucose because the COD of a MM is basically zero.

The pro?les of the ef?uent COD and the COD removal at different oil-to-glucose ratios are shown in Fig.1.At all studied oil-to-glucose ratios,except at10:1,both COD and COD removal tended to remain unchanged throughout the entire operation time.The highest COD removal,around85%,was observed at an oil-to-glucose ratio of 40:1.At an oil-to-glucose ratio of10:1,although the highest COD removal was achieved at the beginning of the SBR operation,it became lower when the operation time was longer than8h.From the results of the ef?uent COD and COD removal,the steady-state

188S.Pansiripat et al./Biochemical Engineering Journal 49 (2010) 185–191

Table 1

Characterization of the feed solutions with various oil-to-glucose ratios for the SBRs at a constant OLR of 2kg/m 3day and a constant cycle time of 2days/cycle.Parameters

Oil-to-glucose ratio Without

60:140:130:120:110:1Chemical oxygen demand (mg/l)33,29033,39033,43033,48033,57033,850Total organic carbon (mg/l)3,7903,8303,8503,8703,9104,030Total nitrogen (mg/l)237239241242244252Total phosphorus (mg/l)271274275277279288C:N ratio 16:116:116:116:116:116:1C:P ratio

14:114:114:114:114:114:1Palm oil concentration (mg/l)6,000

6,0006,0006,0006,0006,000Glucose concentration (mg/l)01,0001,0052,0003,0006,000Surface tension (mN/m)

71.7

71.5

71.9

70.9

71.8

operations at all investigated oil-to-glucose ratios were found to be in the range of 8–14days.

The results of surface tension reduction,COD removal,and oil removal of the studied SBR system during the

steady-state opera-tion at different oil-to-glucose ratios are shown in Fig.2.The surface tension reduction was found to statistically increase after the glu-cose addition,indicating that the added glucose enhanced the biosurfactant production in the reactor.However,an increase in the glucose concentration did not further increase the surface tension reduction.This implies that the biosurfactant concentration in the SBR units perhaps reached or exceeded its CMC,so the surface ten-sion remained steady at a particular value (28–30mN/m),resulting in a constant surface tension reduction percentage.According to the

Fig.1.Process performance of the studied SBR units at different oil-to-glucose ratios,a constant OLR of 2kg/m 3day,and a constant cycle time of 2days/cycle:(a)ef?uent COD and (b)COD removal.

works of Cassidy [25]and Cassidy et al.[26],although the concen-tration of the typical metabolic products was more than 10%,the surface tension of the culture medium was not below 30mN/m.In the present work,the minimum surface tension was lower than 30mN/m,thus implying that a large quantity of surface-active species was excreted in the SBR reactors.The obtained minimum surface tension is also consistent with a characteristic of glycolipid biosurfactants produced by Pseudomonas species—the rhamnolipid compounds [6,27].It was previously reported that P.aeruginosa SP4excreted a mixture of eleven rhamnolipids when grown in a shaking incubator (batch fermentation)using a nutrient broth with palm oil as a carbon source [23].Thus,it is expected that the rham-nolipid biosurfactant was also produced by the strain SP4in the SBR units.However,a rhamnolipid component in the biosurfactant obtained from the SBR system could differ from that previous study because of the different substrate composition and speci?c culture conditions.

In contrast to the surface tension reduction,both COD removal and oil removal ?rst increased with increasing glucose concentra-tion.The highest COD removal,85.1%,and the highest oil removal,77.7%,were achieved at an oil-to-glucose ratio of 40:1.A further increase in the glucose concentration (oil-to-glucose ratios of 30:1,

20:1,and 10:1)slightly reduced the COD removal while strongly decreasing the oil removal.The results indicate that the addition of glucose to the culture medium at a high concentration can limit the palm oil uptake by the strain SP4,perhaps because the microbe con-sumed the added glucose instead of the palm oil.From the results of the microbial cell concentration,in terms of MLSS,and the cell wash-out,in terms of SS,an increase in the glucose concentration in the feed solution signi?cantly increases both (as shown in Fig.3),

Fig.2.Process performance of the studied SBR units during steady-state operation at different oil-to-glucose ratios,a constant OLR of 2kg/m 3day,and a constant cycle time of 2days/cycle in terms of surface tension reduction,COD removal,and oil removal.

S.Pansiripat et al./Biochemical Engineering Journal 49 (2010) 185–191

189

Fig.3.MLSS and SS at different oil-to-glucose ratios during steady-state operation and a constant OLR of 2kg/m 3day and a constant cycle time of 2days/cycle.

indicating that the addition of glucose to the culture medium can enhance both the SBR performance and the process stability,up to certain limits,and the biosurfactant production was optimized at the oil-to-glucose ratio of 40:1.

3.2.Biosurfactant production and concentration

After the SBR units were operated at the optimum oil-to-glucose ratio of 40:1to reach the steady-state condition,the surface ten-sions of the ef?uent samples taken during the aeration step were measured in order to investigate the pro?le of the biosurfactant concentration in the SBRs.Fig.4shows the surface tension pro?le of the ef?uent as a function of aeration time during steady-state operation at an OLR of 2kg/m 3day,a cycle time of 2days/cycle,and an oil-to-glucose ratio of 40:1.The surface tension of the ef?u-ent was slightly reduced during the ?rst 4h of the aeration period,which can be attributed to the time taken for the bacterial culture to acclimate to the sudden organic loading.At an aeration time of 6h,the surface tension of the ef?uent was strongly decreased from 66to 35mN/m,implying that a large quantity of the biosurfactant was excreted by the strain SP4.When the aeration time was longer than 10h and up to the end of the aeration step,the surface tension of the ef?uent was slightly reduced before remaining unchanged,in the range of 28–30mN/m.The results suggest that the biosurfac-tant concentration in the SBRs either reached or exceeded its CMC when the aeration step was longer than 10

Fig.4.Surface tension as a function of aeration time at an oil-to-glucose ratio of 40:1,an OLR of 2kg/m 3day,and a cycle time of 2

days/cycle.

Fig.5.Surface tension of ef?uent taken at different aeration times as a function of dilution ratio when the SBR units were operated at an oil-to-glucose ratio of 40:1,an OLR of 2kg/m 3day,and a cycle time of 2days/cycle.

Next,the biosurfactant concentration in the reactor was esti-mated by using the CMD technique.Fig.5shows the surface tension as a function of dilution ratio of the samples taken from the reac-tor at different aeration times.The surface tension of all the test samples,except for the samples taken at aeration times of 42and 48h,was higher than 30mN/m (the minimum surface ten-sion)when they were diluted to 0.95times (dilution ratio of 95:5),indicating that the biosurfactant concentration was lower than its CMC.The results also show that the samples taken at an aeration time of 42and 48h had the highest surface activity,compared to the others.The surface tensions of these two samples rose above 30mN/m when they were diluted to 1.1times (dilution ratio of 75:25).Hence,the biosurfactant concentration in the SBRs was maximized at an aeration time of 42h,and the highest biosurfac-tant concentration of 1.1×CMC could be retained until the end of the aeration step—an aeration time of 48h.Although it was pre-viously reported that the maximum biosurfactant concentration in the SBRs could be obtained at 70×CMC,the produced biosur-factant in those studies was totally biodegraded by the end of the operating cycle,leading to an increase in the surface tension and a zero CMD [3,4].Moreover,the high biosurfactant concentration in the bioreactor can cause the excessive foam production,which subsequently decreases the process performance due to the loss of microbial cells from the system.To minimize the foam forma-tion,several operational parameters,such as pH and aeration rate,have to be further optimized [28].In some cases,mechanical foam breakers and chemical antifoam agents are used,but the addition of chemical antifoam agents can affect downstream processing [29],microbial cell metabolism [1,30],and oxygen transfer rate [31].In the present work,the investigated SBR system was operated to produce the biosurfactant without the foaming problem.Although the biosurfactant concentration in the SBR system was quite low,the biosurfactant product can be enriched using several existing processes,including the foam fractionation technique [32].3.3.Rhamnolipid composition in the biosurfactant product

To analyze the rhamnolipid composition,the biosurfactant in the ef?uent sample obtained from the SBRs operating under the optimum conditions was extracted with a CH 3Cl–C 2H 5OH mixture before being subjected to the HPLC.It has been reported that P.aeruginosa SP4produces rhamnolipid biosurfactants when grown in a shaking incubator (batch fermentation)using a nutrient broth supplemented with palm oil as the carbon source [23].In that study,

190S.Pansiripat et al./Biochemical Engineering Journal

49 (2010) 185–191

Fig.6.Surface tension versus concentration of the biosurfactant extracted from the ef?uent sample taken from the SBRs operated at an oil-to-glucose ratio of40:1,an OLR of2kg/m3day,and a cycle time of2days/cycle.

the biosurfactant was also extracted from the liquid culture with the same solvent mixture.After being analyzed by HPLC,six high-purity rhamnolipid-containing fractions–called A,B,C,D,E,and F–were obtained,and based on the peak areas,the biosurfactant was composed of fractions A,B,C,D,E,and F at0.7%,1.5%,73.5%,9.5%, 13.3%,and1.4%,respectively.The chemical structure of the most abundant fraction(fraction C)was identi?ed as Rha-C10-C10,while A,B,D,E,and F were characterized as Rha-Rha-C8-C10,Rha-C8-C10, Rha-C10-C12:1,Rha-C10-C12,and Rha-Rha-C10-C14:1,respectively, with small contributions from their isomers.In the present work, the biosurfactant produced by the strain SP4using the SBR process contained fractions A,C,and F at65.1%,6.2%,and4.9%,respectively, while fractions B,D,and E could not be detected.It is known that the predominant rhamnolipid species in the excreted biosurfactant could be in?uenced by the substrate composition[11],the speci?c culture conditions[11],the age of the culture[33],and the cho-sen Pseudomonas strain[33].For example,Déziel et al.[33]used mannitol for the biosurfactant production by P.aeruginosa57RP and found that Rha-Rha-C10-C10was the major component in the biosurfactant product.However,when naphthalene was used as a carbon source instead of mannitol,the most abundant rhamnolipid became Rha-Rha-C10.Nitschke et al.[10]also found that the main rhamnolipid in the biosurfactant produced by P.aeruginosa LBI was Rha-Rha-C10-C10when glucose or glycerol was used.In contrast, the biosurfactant produced by using spent soybean oil,chicken fat, or soybean oil soapstock as a carbon source predominantly con-tained Rha-C10-C10[10].Therefore,the rhamnolipid composition of the biosurfactant produced by P.aeruginosa SP4in this present study(using the MM with palm oil and glucose in the continuous SBRs)differed from that in the previous study(using the nutrient broth with palm oil in batch fermentation),perhaps because of the different substrate compositions and speci?c culture conditions.

3.4.Surface activities of the biosurfactant product

The surface activities of the extracted biosurfactant were stud-ied in terms of surface tension reduction and CMC.A plot of surface tension versus initial concentration of the biosurfactant extracted from the ef?uent sample taken from the SBR units operated under the optimum conditions is shown in Fig.6.The surface tension of pure water rapidly decreased as the biosurfactant concentra-tion increased,and a minimum surface tension of about30mN/m was observed at an initial biosurfactant concentration greater than 150mg/l.From the break point of the surface tension versus its log of concentration curve,the CMC of the biosurfactant product was found to be150mg/l.The obtained value of the minimum sur-face tension and CMC is very much in accord with the previously reported value of rhamnolipid compounds[6,13,25,34–36].

The surface-active properties of each rhamnolipid homologue are governed by the presence of unsaturated bonds,the length of alkyl chains,and the size of the hydrophilic head group[6].For example,the CMC of Rha-Rha-C10-C10was about5mg/l,while Rha-C10-C10showed a CMC of40mg/l[37].The CMCs of the more hydrophilic rhamnolipid species,including Rha-Rha-C10and Rha-C10,were200mg/l[34].Rhamnolipids containing unsaturated hydrophobic chains showed higher CMCs,compared to the cor-responding saturated forms[38],and the presence of larger fatty acid chains decreased the CMCs of the rhamnolipids[13].However, because of the dif?culty in separating the rhamnolipid components into a single homologue,the biosurfactant product is usually char-acterized as a mixed biosurfactant system instead.Generally,the biosurfactants produced by P.aeruginosa strains can reduce the sur-face tension of pure water to below30mN/m with a CMC in the range of5–200mg/l,depending on the ratio and composition of the rhamnolipid species[6,25,35].Mata-Sandoval et al.[39]found that a biosurfactant produced by P.aeruginosa UG2,with a high con-tent of Rha-Rha-C10-C12and Rha-Rha-C10-C12:1,showed a CMC of 37mg/l,while the CMC of that rich in Rha-Rha-C10-C10was53mg/l. It was previously reported that the biosurfactant produced by P. aeruginosa SP4using a batch fermentation showed a CMC of about 200mg/l[23].The higher CMC of the SP4biosurfactant in that pre-vious work should correspond to a difference in the rhamnolipid components.

4.Conclusions

P.aeruginosa SP4,isolated from petroleum-contaminated soil in Thailand,was used to produce a biosurfactant from palm oil in a MM supplemented with glucose.The biosurfactant production was done in two identical lab-scale aerobic SBR units.The results indi-cated that glucose slightly affected the biosurfactant production but strongly enhanced the microbial growth in the studied SBR units. Our?ndings also showed that the selected carbon source played a key role on the components in the biosurfactant product. Acknowledgements

This work was?nancially supported by The Research Unit of Applied Surfactants for Separation and Pollution Control,under The Ratchadapisek Somphot Fund,Chulalongkorn University.The Royal Golden Jubilee Ph.D.scholarship for Miss Orathai Pornsun-thorntawee is also acknowledged.

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