Production and stability studies of the biosurfactant isolated from marine Nocardiopsis sp. B4

Desalination 285 (2012) 198–204

Contents lists available at SciVerse ScienceDirect

Desalination journal homepage: www.elsevier.com/locate/desal

Production and stability studies of the biosurfactant isolated from marine Nocardiopsis sp. B4 A. Khopade a, R. Biao b, X. Liu b, K. Mahadik a, L. Zhang b, C. Kokare a, c,⁎ a b c

Department of Pharmaceutical Biotechnology, Poona College of Pharmacy, Bharati Vidyapeeth Deemed University, Pune, 411 038, India Chinese Academy of Science Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China Department of Pharmaceutics, STES, Sinhgad Institute of Pharmacy, Narhe, Pune, 411 041, India

a r t i c l e

i n f o

Article history: Received 7 June 2011 Received in revised form 21 September 2011 Accepted 4 October 2011 Available online 9 November 2011 Keywords: Biosurfactant Nocardiopsis Optimization Stability Actinomycetes

a b s t r a c t A potential biosurfactant producing strain, marine Nocardiopsis B4 was isolated from the West coast of India. Culture conditions involving variations in carbon and nitrogen sources were examined at constant pH, temperature and revolutions per min (rpm), with the aim of increasing productivity in the process. The biosurfactant production was followed by measuring the surface tension, emulsification assay and emulsifying index E24. Enhanced biosurfactant production was carried out using olive oil as the carbon source and phenyl alanine as the nitrogen source. The maximum production of the biosurfactant by Nocardiopsis occurred at a C/ N ratio of 2:1 and the optimized bioprocess condition was pH 7.0, temperature 30° C and salt concentration 3%. The production of the biosurfactant was growth dependent. The surface tension was reduced up to 29 mN/m as well as the emulsification index E24 was 80% in 6 to 9 days. Properties of the biosurfactant that was separated by acid precipitation were investigated. The biosurfactant activity was stable at high temperature, a wide range of pH and salt concentrations thus, indicating its application in bioremediation, food, pharmaceutical and cosmetics industries. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Research in the area of biosurfactants has expanded in recent years due to its potential use in different areas, such as the food industry, agriculture, pharmaceuticals, oil industry, petro chemistry, paper and pulp industry. The development of this line of research is of paramount importance, mainly in view of the present concern regarding the protection of the environment. They are not only useful as antibacterial, antifungal and antiviral agents, but also have potential for use as major immunomodulatory molecules, anti-adhesive agents and even in vaccines and gene therapy [1–4]. Involvement of biosurfactants in microbial adhesion and desorption has been widely described. For example, inhibition of uropathogen biofilm formation on silicone rubber by protein-like biosurfactants obtained from Lactobacillus fermentum RC-14 was reported [1]. Also, exposure to suspensions of active probiotics and the consumption of buttermilk containing Lactococcus lactis 53 were reported to influence the biofilm formation on silicone rubber voice prostheses [1], possibly due to the release of biosurfactants. Biosurfactants, are produced by bacteria or yeast from various substrates including sugars, glycerol, oils, hydrocarbons and agricultural wastes. Biosurfactants are

⁎ Corresponding author at: Department of Pharmaceutics, STES, Sinhgad Institute of Pharmacy, Narhe, Pune, 411 041, India. Tel.: + 91 20 66831806; fax: + 91 20 24699051. E-mail address: [email protected] (C. Kokare). 0011-9164/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.desal.2011.10.002

classified as glycolipids, lipopeptides, phospholipids, fatty acids, neutral lipids, and polymeric or particulate compounds [2,5]. The hydrophobic portion of the molecule is long-chain fatty acids, hydroxyl fatty acids or a-alkyl-b-hydroxyl fatty acids. The hydrophilic moiety can be a carbohydrate, amino acid, cyclic peptide, phosphate, carboxylic acid or alcohol. Biosurfactants have been receiving increasing attention as a result of their unique properties, i.e. mild production conditions, lower toxicity and higher biodegradability, compared to their synthetic chemical counterparts [3]. Even though interest in biosurfactants is increasing, these compounds do not compete economically with synthetic surfactants. To reduce production costs, different routes could be investigated such as the increase of yields and product accumulation; the development of economical engineering processes and the use of cost-free or cost credit feedstock for microorganism growth and surfactant production. The choice of inexpensive raw materials is important to overall economy of the process because they account for 50% of the final product cost and also reduce the expenses with wastes treatment [1,6,7]. Glycolipid from Rhodococcus erythropolis, R. aurantiacus and surface active lipid from Nocardia erythropolis were studied in the literature survey. There are very few reports on biosurfactants production from marine actinomycetes [6]. The objective of present study was to isolate marine actinomycetes and the identification of strain for potent biosurfactant production. The cultural conditions for maximum production of biosurfactant and the stability study of the product were investigated.

A. Khopade et al. / Desalination 285 (2012) 198–204

2. Material and methods

199

tension and emulsification assay of supernatant samples obtained after cell separation [18,19].

2.1. Isolation of actinomycetes strain B4 Marine sediment sample was collected from Mumbai coastal region of India at the time of low tide [8]. Collected sediment sample was suspended in sterile water and mixed on rotary incubator shaker (New Brunswick Scientific, Model Excella E24, USA), at 150 rpm for 20 min. Different marine actinomycete species were isolated by using selective media such as glycerol yeast extract agar, starch casein agar, maltose yeast extract agar and glucose aspargine agar (Himedia, India). The isolated strains were screened for biosurfactant production by using different techniques [9–14]. Maximum biosurfactant producing marine sp. B4 was maintained on glycerol yeast extract agar medium [15]. 2.2. Identification of strain B4 Identification of strain was done by scanning electron microscopy (SEM) (Pune, India), 16S r-DNA sequencing, biochemical and cultural characterizations. The method adopted for preparation of slide culture for SEM analysis was used, as described by Williams and Davies [12,15–17]. 2.3. Inoculum preparation and culture condition The glycerol yeast extract medium prepared in artificial seawater (ASW) was used for development of inoculum. The seed culture was prepared in 100 ml conical flasks containing 50 ml of medium by inoculating 2.0 ml of spore suspension containing 2.5 to 3.0 × 10 6 CFU ml − 1 and cultivated with agitation (150 rpm) at 28 °C for 4 days. The seed culture (50 ml) was inoculated in the 1 l fermentation medium prepared in artificial sea water supplemented with 0.1 ml trace element solution. The pH of the medium was adjusted to 7.0. Fermentation was carried out in 2 l bench scale fermenter (New Brunswick Scientific, USA) for 12 days. The operating conditions during batch fermentation were temperature 28 °C, agitation rate 150 rpm and the aeration rate 1.0 VVM [8]. 2.4. Medium optimization The medium optimization was conducted in a series of experiments changing one variable at a time, keeping other factors unchanged. The production of biosurfactant was growth dependent. Cell growth and the accumulation of metabolic products were strongly influenced by medium composition such as carbon sources, nitrogen sources. Three factors were chosen aiming to obtain higher productivity of the biosurfactant: carbon source (C), nitrogen source (N) and C/N ratio. The carbon sources used were n-hexadecane (2% w/v) (Himedia, India), olive oil (2% w/v) (commercial type), sucrose (Himedia, India), trehalose (Himedia, India), maltose (Himedia, India), dextrose and glucose (Himedia, India) (20 g/l), with ammonium chloride (NH4Cl) (Himedia, India) as nitrogen source. For evaluation of the most appropriate nitrogen sources for the production of biosurfactants, phenyl alanine (Himedia, India), urea (Himedia, India), ammonium sulfate (Merck, India), NH4Cl and sodium nitrate (NaNO3)(Merck, India), were employed at a concentration of 1 g/l with the optimum carbon source. The C/N ratio (with optimized carbon and nitrogen sources) was varied from 10 to 40 by keeping a constant nitrogen source concentration 1 g/l [18–21]. 2.5. Biosurfactant production kinetics The kinetics of biosurfactant production was followed in batch cultures during 12 days at optimum conditions by measuring surface

2.6. Effect of pH, temperature, sodium chloride and aeration on biosurfactant production and activity In order to evaluate the effect of pH and temperature on the biosurfactant production, the pH of medium was adjusted in the range between 4 and 12 and the temperature was set at 4, 15, 25, 30, 35 40, 45 and 60 °C. The pH of the medium was measured with a digital pH-meter (Systronics, India). To examine the effect of sodium chloride on biosurfactant production in optimized medium, the sodium chloride was added in medium to achieve final concentrations of 1–10% (w/v). Effect of aeration on production of biosurfactant was detected by incubating inoculated fermentation media at different aeration conditions such as 50, 75, 100, 125, 150, 175, 200, 225 and 300 rpm. Biosurfactant production was measured by emulsification assay and absorbance was measured at 400 nm [21]. 2.7. Effect of oils, surfactants and hydrocarbon on biosurfactant production The effect of crude oil and surfactant was evaluated for biosurfactant production. The different oils were used such as castor oil, codliver oil, eucalyptus oil, sesame oil, mustard oil and surfactants such as ethylene diamine tetra acetic acid (EDTA) (Himedia, India), cetyl trimethyl ammonium bromide (CTAB) (Himedia, India), sodium dodicyl sulfate (SDS) (Loba Chemie, India), tweens 20, 40, 80 (Loba Chemie, India) were added separately in 1% (v/v) and emulsification activity of medium was measured. The hydrocarbons such as diesel, petrol, toluene, xylene, n-hexane and kerosene (commercial grade, India) were added [1% (v/v)] separately in optimized medium and their effect was observed. The surface tension measurement was carried out using the du Nouy ring method [6,8]. 2.8. Surface tension measurement The surface tension measurement of cell free supernatant was determined in a K6 tensiometer, using the du Nouy ring method. The values reported were the mean of three measurements. All measurements were made on cell-free broth obtained by centrifuging the cultures at 10,000 rpm for 20 min [18]. 2.9. Bioemulsifier production assay Actinomycetes species were grown for 12 to 15 days in glycerol yeast extract (GYE) broth. The microbial cells were separated by centrifugation (Eppendorff, model 5810R, Germany) at 10,000 rpm for 15 min at 30 °C. Cell free culture broth (3 ml) was added in 0.5 ml test oil, mixed vigorously for 2 min and incubated at 30 °C for 1 h for phase separation. Aqueous phase was removed carefully and absorbance of aqueous phase was recorded at 400 nm. The absorbance maxima arrived after scanning at entire visible light spectrum. The blank was prepared with sterile medium. An absorbance of 0.01 units at 400 nm multiplied by dilution factor, if any, was considered as one unit of emulsification activity per ml (EU/ml) [6]. 2.10. Emulsification index (E24) Emulsification index of culture samples was determined by adding 2 ml of a hydrocarbon to the same amount of culture, mixing with a vortex for 2 min, and left standing for 24 h. The E24 index is given as percentage of height of emulsified layer (mm) divided by total height of the liquid column (mm) [9–11].

200

A. Khopade et al. / Desalination 285 (2012) 198–204

2.11. Biosurfactant recovery Culture broth was centrifuged at 10,000 rpm for 20 min to get cell free broth. Biosurfactant was precipitated by adjusting the pH of the cell free broth to 2.0 using 6 N hydrochloric acid (HCl) (Merck, India) and keeping it overnight at 4 °C. The precipitate thus formed was collected by centrifugation (10,000 rpm, 20 min; 4 °C) and dissolved in distilled water. Its pH was adjusted to 8.0 with 1 N sodium hydroxide (NaOH) (Qualigens, India), and the solution was lyophilized [18]. 2.12. Stability studies To determine the thermal stability of the biosurfactant, cell-free broth was maintained at a constant temperature range of 20–100 °C for 15 min, then cooled to room temperature and activity of the biosurfactant was investigated. To determine the effect of pH on activity, the pH of the cell free broth was adjusted to different values using 1 N NaOH or 1 N HCl. The effect of addition of different concentration of NaCl on the activity of the biosurfactant was investigated. The biosurfactant was re-dissolved after purification with distilled water containing the specific concentration of NaCl (0–9%, w/v) [22,23]. 3. Results and discussions The advantages of biosurfactants over synthetic ones include lower toxicity, biodegradability, selectivity, specific activity at extreme temperatures, pH and salinity. In our laboratory, we have isolated the strain of marine Nocardiopsis from sediments. 3.1. Characterization of strain B4 The strain B4 showed good growth in the temperature range 25–45 °C in 7 days on glycerol yeast extract agar medium. Outer surface of colonies was perfectly round initially, but later they developed aerial mycelium that may appear velvety and spore formation started after the 4th day of incubation. Spore chain was long and sporulating hyphae were straight. Spores were oval and warty, appeared like hairy and were 1–2.5 mm in size (Fig. 1A and 1B). Good growth was observed at neutral pH. By morphology, SEM and 16S DNA sequencing (Fig. 2), the isolated strain was found to be a member of Nocardiopsis genus [8].

Fig. 1. Scanning electron microscopy of strain B4 [A] × 3000 magnification, and [B] × 6000 magnification.

the surfactant under these conditions. Olive oil was the best carbon source for biosurfactant synthesis. The isolated biosurfactant decreased the surface tension to 30 mN/m and the emulsifying activity was 80%. Similar results were found with biosurfactant production form P. aeruginosa 44T1 [24,25].

3.3. Optimization of cultivation medium

3.3.2. Effect of nitrogen source The effect of nitrogen source affects the biosurfactant production as shown in Fig. 4B. Nocardiopsis sp. was able to use nitrogen sources such as ammonia and urea for biosurfactant production. However, in order to obtain high concentrations of biosurfactant it is necessary to have restrained conditions of these macro-nutrients. Phenyl alanine was the best source of nitrogen for growth and biosurfactant synthesis. Ammonium salts in the form of ammonium chloride were used for growth but not for biosurfactant production and caused a significant decrease in pH (4.03) [24,26,27]. The maximum emulsifying activity and minimal surface tension (30 mN/m) were reached in media with phenyl alanine. No significant change in pH was observed in this case. A similar result was reported in biosurfactant isolated from Pseudomonas fluorescens by Abouseoud et al. [18].

3.3.1. Effect of carbon source The production of biosurfactant was studied by using carbon sources such as n-hexadecane, olive oil, trehalose, sucrose, fructose, maltose and glucose (Fig. 4A). The use of vegetable oil as carbon sources to produce biosurfactants seems to be an interesting and low cost alternative [18]. Screening of nutrient substrates showed that Nocardiopsis supported growth on all substrates although the yield was limited with galactose or starch as carbon sources because of inhibition due to the decrease in pH is probably caused by the production of secondary acid such as uronic acid [18]. The maximum biosurfactant production was occurring only with trehalose, hexadecane and olive oil. The strain grew on starch but did not produce

3.3.3. Effect of carbon/nitrogen ratio The fundamental aspect to the improvement of biosurfactant productivity was the ratio of C/N. These results were obtained using olive oil and phenyl alanine as carbon and nitrogen source respectively since the best results were attained with lower values of this parameter (C/N = 20 ) (ST = 30; EA = 265 U/ml) (Fig. 4C). There were no significant differences between C/N ratios of 30, 35 and 40 in relation to emulsification index, but a C/N ratio of 20 presented a significant difference in relation to the emulsification index. These results are similar with those found using waste frying oil and sodium nitrate as carbon and nitrogen sources respectively [25–28]. Guerra-Santos et al. [29] observed that biosurfactant production was poor with

3.2. Growth characteristics and biosurfactant production from strain B4 Most of the actinomycetes species are slow-growing. Biosurfactant production started in early log phase but there was drastic increase in production at late growth phase and early stationary phase; and biosurfactant production continued up to late stationary phase and after that it declined (Fig. 3). It clearly indicates that biosurfactant production was dependent on the growth phase [8].

A. Khopade et al. / Desalination 285 (2012) 198–204

201

T 99 Nocardiopsis exhalans ES10.1 (AY036000)

Nocardiopsis valliformis 20028T (AY336503) Nocardiopsis metallicus KBS6 T(AJ420769) Nocardiopsis ganjiahuensis HBUM 20038 T(AY336513) Nocardiopsis prasina DSM 43845 T(X97884)

76

Nocardiopsis listeri DSM40297 T(X97887) 76

76

Nocardiopsis alkaliphila DSM 44657 T(AY230848)

B4 95

Nocardiopsis alba DSM 43377 T(X97883) Nocardiopsis umidischolae 66/93 T(AY036001) 100

Nocardiopsis tropica VKM Ac -1457 T(AF105971) Marinactinospora thermotoleransSCSIO 00652 T(EU698029)

0.005

Fig. 2. Neighbor-joining phylogenetic tree of strain B4 made by MEGA 4.0. Numbers at nodes indicate levels of bootstrap support (%) based on a neighbor-joining analysis of 1000 resampled datasets; only values >50% are given. NCBI accession numbers are given in parentheses. Bar, 0.005 nucleotide substitutions per site.

both yeast extract and nitrate as nitrogen sources. When the yeast extract was omitted, the biomass concentration decreased, rhamnolipid increased and a moderate accumulation of glucose occurred, indicating a nitrogen-limiting medium. 3.4. Kinetics of biosurfactant production The biosurfactant production and surface tension were dependent on growth of culture in the fermentation medium. The surface tension dropped rapidly after inoculation, reaching its lowest value (29 mN/m) during exponential phase after about 9 days of growth (Fig. 3A and Fig. 3B). The emulsification activity plot, a measure of biosurfactant concentration, showed that insufficient surfactant was initially present to form micelles. At about 6th day of growth, the surfactant concentration started to increase, reaching its maximum after about 9th day. The increase in surface tension and the decrease in E24 after 12th of incubation showed that biosurfactant biosynthesis stopped and is probably due to the production of secondary metabolites which could interfere with emulsion formation and the adsorption of surfactant molecules at the oil–water interface [30]. These results indicate that the biosurfactant biosynthesis from olive oil occurred predominantly during the exponential growth phase, suggesting that the biosurfactant is produced as primary metabolite accompanying cellular biomass formation (growth-associated kinetics) [31]. This property suggests that biosurfactant could be effectively produced under chemostat conditions or by immobilized cells [18,19,32,33]. 3.5. Effect of sodium chloride, pH, temperature and aeration on biosurfactant production The strain B4 was found to be moderately halophilic in nature as maximum biosurfactant production was obtained in presence of 3%

Fig. 3. Growth kinetics and biosurfactant production of Nocardiopsis B4 sp. (OD600).

(w/v) of NaCl and it retained almost 80% of its activity in presence of 12% (w/v) of NaCl. (Fig. 5A). The strain B4 showed gradual increase in biosurfactant production and optimum pH for biosurfactant production was found to be 7 (Fig. 5B). The research was focused on the isolation of alkaline biosurfactant from microbes because there is tremendous potentiality of biosurfactant in detergent industry. The strain B4 showed good growth in the temperature range of 25–45 °C but optimum growth was observed at 30 °C (Fig. 5C). This clearly indicates the moderately thermostable nature of the biosurfactant. The maximum biosurfactant production was obtained at 150 rpm. Until today, bacteria belonging to genus Bacillus have been exploited for commercial production of biosurfactant [21,24]. There is no such report on isolation of moderately thermostable surfactant from marine Nocardiopsis sp. 3.6. Effect of oils, surfactants and hydrocarbons on production of biosurfactant Fermentation was carried out with addition of different concentrations of oils, surfactant and hydrocarbons in the fermentation medium. It was observed that olive oil, tween 80 and hexane as a substrate showed maximum activity against all test oils, surfactants and hydrocarbons respectively. Olive oil and tween 80 showed emulsification activity at 198 EU/ml and 225 EU/ml respectively (Fig. 6A and 6B). Hexane was used [6] as a substrate for biosurfactant production and it was observed that 1% v/v showed maximum biosurfactant production activity (Fig. 6C). 3.7. Stability study 3.7.1. Temperature stability The applicability of biosurfactants in several fields depends on their stability at different temperatures and pH values. The stability of biosurfactant was tested over a wide range of temperature. The biosurfactant produced by Nocardiopsis sp. was shown to be thermostable (Fig. 7A). Heating of the biosurfactant to 100 °C caused no significant effect on the biosurfactant performance. The emulsification activity was quite stable at the temperatures used (E24 = 66%) in comparison with synthetic surfactants such as SDS which exhibits a significant loss of emulsification activity beginning at 70 °C [31]. Therefore, it can be concluded that this biosurfactant maintains its surface properties unaffected in the range of temperatures between 30 and 100 °C. This activity was discovered indicating the usefulness of the biosurfactant in food, pharmaceutical and cosmetics industries where heating to achieve sterility is of paramount importance [18,25].

Surface tension (mN/m)

60 40 20

0 1

G

2

3

4

Carbon sources

B 80

Surface tension (mN/m)

60

40

20

8

9

10

60

40

20

at tr

11

12

45

60

9

10

4

di So

6

ni

0

um

sp A

ch o. m

m A

7

e

ne

a ar

gi

re U

rid

e

ne

lo

la

ni

ni A

la la ny

P

A

m

m

he

o.

su

lp

ha

te

ne

0

6

80

5

Surface tension (mN/m)

B

5

NaCl (% w/v)

8

O

l iv e al o il at D os ex e tr os e S tr M ac an h n S itol uc Tr ro e h se a H ex lo s ad e e Fr c n uc e to M se al to se

0

7

E24 (%)

80

60

A

40

A 100

80

A. Khopade et al. / Desalination 285 (2012) 198–204

20

202

pH

Nitrogen source

C 100

300 Emlusification activity Surface tension 200

100

Surface tension (mN/m)

80

60

40

20

30

35

40

C:N ratio Fig. 4. [A] Effect of carbon source on biosurfactant production, [B] effect of nitrogen source on biosurfactant production, and [C] effect of C:N ratio on biosurfactant production.

3.7.2. pH stability The surface activity of the crude biosurfactant remained relatively stable to pH changes between pH 8 and 12, showing higher stability at alkaline pH 9 than acidic conditions. At pH 12, the value in emulsification activity (E24) showed almost 66% activity, whereas below pH 7 activity was decreased up to 55%. In addition, for pH values lower than 6, the samples become turbid, due to partial precipitation of the biosurfactant. Fig. 7B shows the effect of pH on the biosurfactant properties. These results indicate that increase pH has a positive effect on emulsification activity and emulsion stability. This could be caused by a better stability of fatty acid surfactant micelles in the presence of NaOH and the precipitation of secondary metabolites at

40

25

35

20

30

15

25

10

15

0

0

4

Surface tension (mN/m) Emulsification activity (EU/ml)

C

Temperature (°C) Fig. 5. [A] Effect of NaCl on biosurfactant production, [B] effect of pH on biosurfactant production, and [C] effect of temperature on biosurfactant production.

higher pH values. The effect of pH on surface activity has been reported for biosurfactants for different microorganisms [18,34]. 3.7.3. Effect of salinity The effect of sodium chloride addition on biosurfactant produced from Nocardiopsis was studied. Optimum stability of biosurfactant was observed at 3% NaCl concentration. Little changes were observed in increased concentration of NaCl up to 8% (w/v) (Fig. 7C). At higher concentration of NaCl the biosurfactant retains 50% of the emulsification activity. The biosurfactant has stability at alkaline pH and high salinity; such a biosurfactant may be useful for bioremediation of spills in marine environment because of its stability in alkaline condition and in the presence of salt. Stability of emulsion in the presence

A. Khopade et al. / Desalination 285 (2012) 198–204

A

200 100 150 100

50

50

Se n

0 10

90

80

70

60

50

30

ro il am om oi l

10

11

12

7

8

9

0 9

100

6

50

4

200

8

100

7

E24 (%)

300

6

oi l

liv e

ut

od C

Ec u

C

oc

C

on

lo v

tu

e

s

oi l

oi l

oi l al yp

il ro

O

liv e

te as C

Temperature (°C)

B 150

Oils

pH

150

X-

10

0

80 n

C

Tr ito n

ee Tw

ee Tw

Tw

ee

n

n

40

20

S SD

TA C

ED

B

0 TA

Emulsification activity (EU/ml)

40

0

0

Surfactants

E24 (%)

100

60

50

40

5

20

4

0 1

Surface tension (mN/m)

80

3

C

2

B

150

E24 (%)

Emulsification activity (EU/ml)

250

5

A

203

NaCl (% w/v) Fig. 7. Effect of [A] temperature, [B] pH and [C] salinity on biosurfactant stability.

l ie se D

tr

ol

s

Pe

K er

os

en

ne Xy le

an H ex n

To

lu

ne

e

0

Hydrocarbons Fig. 6. [A] Effect of oils on biosurfactant production, [B] effect of surfactants on biosurfactant production, and [C] effect of hydrocarbons on biosurfactant production.

of salt has been reported as one of the properties of the biosurfactant produced by Bacillus licheniformis strain JF-2 [21]. 4. Conclusions In the present study the biosurfactant isolated from marine Nocardiopsis sp. showed good stability at high temperature, a wide range of pH and salt concentrations and the maximum biosurfactant production was observed with olive oil as a carbon source and phenyl alanine as the nitrogen source. The important finding was thermo stability of biosurfactant; isolated biosurfactant was extreme stability

at high temperature (100°). The thermal stability of the biosurfactants increases its scope of application in a broader perspective including at conditions where high temperatures prevail as in microbial enhanced oil recovery. Considering the potential need of halotolerant strains and biosurfactants for the bioremediation of oil contaminated sites (oil spills), it is mandatory to screen and develop potential biosurfactant producers from the marine environment. It was found that the biosurfactant produced by the marine Nocardiopsis was stable up to 8% NaCl; however the chemical surfactants are deactivated by 2–3% salt concentration. The biosurfactant was isolated from natural sources thus, indicating the application of the biosurfactant in food, pharmaceutical and cosmetics industries. Acknowledgments The authors would like to acknowledge All India Council for Technical Education (AICTE), HRD Ministry, New Delhi, Govt. of India for

204

A. Khopade et al. / Desalination 285 (2012) 198–204

financial support to this research project under National Doctoral Fellowship (NDF), 2010–2013. References [1] L.R. Rodrigues, J.A. Teixeira, H.C. Vander Mei, R. Oliveira, Physicochemical and functional characterization of a biosurfactant produced by Lactococcus lactis 53, Colloids Surf., B 49 (2006) 79–86. [2] J.D. Desai, I.M. Banat, Microbial production of surfactants and their commercial potential, Microbiol. Mol. Biol. Rev. 61 (1997) 47–64. [3] E.Z. Ron, E. Rosenberg, A review of natural roles of biosurfactants, Environ. Microbiol. 3 (2001) 229–236. [4] I.M. Banat, R.S. Markkar, S.S. Cameotra, Potential commercial applications of microbial surfactants, Appl. Microbiol. Biotechnol. 53 (2000) 495–508. [5] S. Mukherjee, P. Das, R. Sen, Towards commercial production of microbial surfactants, Trends Biotechnol. 24 (2006) 509–515. [6] C.R. Kokare, S.S. Kadam, K.R. Mahadik, B.A. Chopade, Studies on bioemulsifier production from marine Streptomyces sp. S1, Ind. J Biotech. 6 (2007) 78–84. [7] S. Maneerat, K. Phetrong, K. Song, Isolation of biosurfactant-producing marine bacteria and characteristics of selected biosurfactant, J. Sci. Technol. 29 (2007) 781–791. [8] S. Chakraborty, A. Khopade, C. Kokare, K. Mahadik, B. Chopade, Isolation and characterization of novel α-amylase from marine Streptomyces sp. D1, J. Mol. Catal. B: Enzym. 58 (2009) 17–23. [9] T.A.A. Moussa, G.M. Ahmed, S.M.S. Abdel-hamid, Optimization of cultural conditions for biosurfactant production from Nocardia amarae, J. Appl. Sci. Res. 2 (11) (2006) 844–850. [10] A.S. Kumar, K. Mody, B. Jha, Evaluation of biosurfactant/bioemulsifier production by a marine bacterium, Bull. Environ. Contam. Toxicol. 79 (2007) 617–621. [11] C.D. Cunha, M. do Rosário, A.S. Rosado, S.G.F. Leite, Serratia sp. SVGG16: a promising biosurfactant producer isolated from tropical soil during growth with ethanol-blended gasoline, Process. Biochem. 39 (2004) 2277–2282. [12] F.M. Bento, F.A. Oliveira Camargo, B.C. Okekeb, W.T. Frankenberger, Diversity of biosurfactant producing microorganisms isolated from soils contaminated with diesel oil, Microbiol. Res. 160 (2005) 249–255. [13] S. Joshi, C. Bharucha, S. Jha, S. Yadav, A. Nerurkar, A.J. Desai, Biosurfactant production using molasses and whey under thermophilic conditions, Bioresour. Technol. 99 (2008) 195–199. [14] G.S. Kiran, T.A. Thomas, J. Selvin, Production of a new glycolipid biosurfactant from marine Nocardiopsis lucentensis MSA04 in solid-state cultivation, Colloids Surf., B 78 (2010) 8–16. [15] S. Chakraborty, A. Khopade, R. Biao, W. Jian, X.Y. Liub, K. Mahadik, B. Chopade, L. Zhang, C. Kokare, Characterization and stability studies on surfactant, detergent and oxidant stable α-amylase from marine haloalkaliphilic Saccharopolyspora sp. A9, J. Mol. Catal. B: Enzym. 68 (2011) 52–58. [16] A. Gnanamania, V. Kavithaa, N. Radhakrishnana, G.S. Rajakumara, G. Sekaranb, A.B. Mandala, Microbial products (biosurfactant and extracellular chromate reductase) of marine microorganism are the potential agents reduce the oxidative stress induced by toxic heavy metals, Colloids Surf., B 79 (2010) 334–339. [17] S.T. Williams, F.L. Davies, Use of scanning electron microscope for the examination of actinomycetes, J. Gen. Microbiol. 48 (1964) 171–177.

[18] M. Abouseoud, R. Maachi, A. Amrane, S. Boudergua, A. Nabi, Evaluation of different carbon and nitrogen sources in production of biosurfactant by Pseudomonas fluorescens, Desalination 223 (2008) 143–151. [19] T.B. Lotfabada, M. Shourianc, R. Roostaazada, A.R. Najafabadi, M.R. Adelzadeha, K.A. Noghabic, An efficient biosurfactant-producing bacterium Pseudomonas aeruginosa MR01, isolated from oil excavation areas in south of Iran, Colloids Surf., B 69 (2009) 183–193. [20] G. Seghal Kiran, T.A. Hema, R. Gandhimathi, J. Selvin, T. Anto Thomas, T.R. Ravji, K. Natarajaseenivasan, Optimization and production of a biosurfactant from the sponge-associated marine fungus Aspergillus ustus MSF3, Colloids Surf., B 73 (2009) 250–256. [21] M.O. Ilori, C.J. Amobi, A.C. Odocha, Factors affecting biosurfactant production by oil degrading Aeromonas spp. isolated from a tropical environment, Chemosphere 61 (2005) 985–992. [22] H. Ghojavand, F. Vahabzadeh, E. Roayaei, A.K. Shahraki, Production and properties of a biosurfactant obtained from a member of the Bacillus subtilis group (PTCC 1696), J. Colloid Interface Sci. 324 (2008) 172–176. [23] E.J. Gudi˜na, J.A. Teixeira, L.R. Rodrigues, Isolation and functional characterization of a biosurfactant produced by Lactobacillus paracasei, Colloids Surf., B 76 (2010) 298–304. [24] L.M. Prieto, M. Michelon, J.F.M. Burkert, S.J. Kalil, C.A.V. Burkert, The production of rhamnolipid by a Pseudomonas aeruginosa strain isolated from a southern coastal zone in Brazil, Chemosphere 71 (2008) 1781–1785. [25] C.N. Mulligan, B.F. Gibbs, Correlation of nitrogen metabolism with biosurfactant production by Pseudomonas aeruginosa, Appl. Environ. Microbiol. 55 (1989) 3016–3019. [26] L.M. Santa Anna, G.V. Sebastian, E.P. Menezes, T.L.M. Alves, A.S. Santos, J.N. Pereira, D.M.G. Freire, Production of biosurfactants from Pseudomonas aeruginosa PA1 isolated in oil environments, Braz. J. Chem. Eng. 19 (2002) 159–166. [27] H. Rashedi, E. Jamshidi, M.M. Assadi, B. Bonakdaepour, Isolation and production of biosurfactant from Pseudomonas aeruginosa isolated from Iranian southern wells oil, Int. J. Environ. Sci. Technol. 2 (2005) 121–127. [28] C.C. Chen, L. Riadi, S.J. Suh, D.E. Ohman, L.K. Ju, Degradation and synthesis kinetics of quorum-sensing autoinducer in Pseudomonas aeruginosa cultivation, J. Biotechnol. 117 (2005) 1–10. [29] L. Guerra-Santos, O. Käppeli, A. Fiechter, Pseudomonas aeruginosa biosurfactant production in continuous culture with glucose as carbon source, Appl. Environ. Microbiol. 48 (1984) 301–305. [30] M. Bonilla, C. Olivaro, M. Corona, A. Vazquez, M. Soubes, Production and characterization of a new bioemulsifier from Pseudomonas putida ML2, J. Appl. Microbiol. 98 (2005) 456–463. [31] A. Persson, E. Österberg, M. Dostalek, Biosurfactant production by Pseudomonas fluorescens 378: growth and product characteristics, Appl. Microbiol. Biotechnol. 29 (1) (1988) 1–4. [32] J. Klein, F. Wagner, Different strategies to optimize the production phase of immobilized cells, Ann. N.Y. Acad. Sci. 501 (1987) 306–316. [33] H. Kim, B. Yoon, C. Lee, H. Suh, H. Oh, T. Katsuragi, Y. Tani, Production and properties of a lipopeptide biosurfactant from Bacillus subtilis C9, J. Ferment. Bioeng. 84 (1) (1997) 41–46. [34] A.S. Abu-Ruwaida, I.M. Banat, S. Haditirto, A. Salem, M. Kadri, Isolation of biosurfactant producing bacteria, product characterization, and evaluation, Acta Biotechnol. 11 (1991) 315–324.