Bioprocess and Biosystems Engineering

, Volume 32, Issue 6, pp 825–835

Production and characterization of lipopeptide biosurfactant by a sponge-associated marine actinomycetes Nocardiopsis alba MSA10

Authors

  • R. Gandhimathi
    • Department of MicrobiologyBharathidasan University
  • G. Seghal Kiran
    • Department of BiotechnologyBharathidasan University
  • T. A. Hema
    • Department of MicrobiologyBharathidasan University
    • Department of MicrobiologyBharathidasan University
  • T. Rajeetha Raviji
    • Department of MicrobiologyBharathidasan University
  • S. Shanmughapriya
    • Department of MicrobiologyBharathidasan University
Original Paper

DOI: 10.1007/s00449-009-0309-x

Cite this article as:
Gandhimathi, R., Seghal Kiran, G., Hema, T.A. et al. Bioprocess Biosyst Eng (2009) 32: 825. doi:10.1007/s00449-009-0309-x

Abstract

A sponge-associated marine actinomycetes Nocardiopsis alba MSA10 was screened and evaluated for the production of biosurfactant. Biosurfactant production was confirmed by conventional screening methods including hemolytic activity, drop collapsing test, oil displacement method, lipase production and emulsification index. The active compound was extracted with three solvents including ethyl acetate, diethyl ether and dichloromethane. The diethyl ether extract was fractionated by TLC and semi-preparative HPLC to isolate the pure compound. In TLC, a single discrete spot was obtained with the Rf 0.60 and it was extrapolated as valine. Based on the chemical characterization, the active compound was partially confirmed as lipopeptide. The optimum production was attained at pH 7, temperature 30°C, and 1% salinity with glucose and peptone supplementation as carbon and nitrogen sources, respectively. Considering the biosurfactant production potential of N. alba, the strain could be developed for large-scale production of lipopeptide biosurfactant.

Keywords

BiosurfactantsLipopeptideMarine actinomycetesOptimizationNocardiopsis

Introduction

Marine sponges (Porifera) have attracted significant attention from various scientific disciplines. Since sponges are efficient filter feeders, any microorganism that can resist the digestive process and immune response can be successfully inhabited in sponges [57]. Bacteria constitute up to 60% of the biomass of sponges [56]. If some compounds are derived from symbiotic microorganisms, it could provide an improved sustainable source of supply for bioactive compound [50]. Reports on new natural products produced by symbiotic microorganisms have recently been increased drastically.

Biosurfactants are attracting attention in recent years because they offer several advantages over chemical surfactants due to its low toxicity, inherent good biodegradability and ecological acceptability [4]. Microorganisms produced a variety of surface active biomolecules called biosurfactants or microbial surfactants. Biosurfactants have unique amphipathic properties, since their complex structure consists of hydrophilic and hydrophobic portion, which enable them to concentrate at interfaces and to reduce the surface tension of aqueous media [12]. According to Rosenberg and Ron [48], biosurfactants are divided into low molecular weight compounds such as glycolipids or lipopeptide and high molecular weight compounds such as polysaccharides, proteins, lipopolysaccharides or lipoproteins. Biosurfactants are found to be secreted in the culture broth or remain adherent to microbial cell surfaces. Several therapeutic and biomedical importance including antibacterial, antifungal, antimycoplasmic, inhibit fibrin clot formation, antitumoral and hemolytic agents are recorded for surface active microorganisms [8, 47, 53].

Although several biosurfactants have been reported from a variety of microorganisms including the most prominent producers, high yielding strains are necessary for the development of novel biosurfactants. Cell growth and the accumulation of metabolic products are strongly influenced by media composition such as carbon, nitrogen sources and growth factors. Thus, the optimization of production environment can result in high yields of metabolites [52]. In the present study, a sponge-associated marine actinomycetes Nocardiopsis alba MSA10 was screened and evaluated as a biosurfactant producer. The cultural conditions were optimized for the production of biosurfactant under submerged fermentation conditions.

Materials and methods

Sample collection and isolation of sponge-associated actinomycetes

Marine sponge Fasciospongia cavernosa was collected from southwest coast of India by SCUBA diving at 10–15 m depth. To avoid cross contamination, only unbroken samples were used for microbiological analysis. The specimens were kept in 1,000 ml of aged sterile seawater (ASW) for 2 h to remove loosely associated microorganisms from inner and outer sponge surfaces. It has been hypothesized that this process may eliminate nonassociated bacteria from the host sponge by digestion. The samples were kept in a sterile incubator oven for 1 h at 40°C to dry the surface, immediately frozen and packed in sterile sip-lap bags. The voucher specimens were stored at −20°C. For the isolation of associated actinomycetes, sponge tissue (1 cm3) was excised from the internal mesohyl area using a pair of sterile scissors. The excised portion was thoroughly washed three times with sterile seawater to remove any bacteria within current canals and then the tissue was homogenized with phosphate buffered saline using a tissue homogenizer. The resultant homogenate was serially diluted with ASW and preincubated at 40°C for 1 h for the activation of dormant cells. The aliquot was plated on various isolation media including marine sponge agar and standard media (Himedia®) such as Emerson agar, actinomycetes agar, actinomycetes isolation agar and marine agar (Table 1). The inoculated plates were incubated at 27°C for 14 days in dark. The morphologically distinct colonies were reisolated and maintained on actinomycetes isolation agar at 4°C.
Table 1

Composition of culture media used for biosurfactant production by N. alba MSA10

Nature of ingredients

AIA medium

MSM medium

Basal medium

ZMB medium

AB medium

Carbon source

Sodium propionate (4.0 g)

Glucose (10.0 g)

Glucose (10.0 g)

Glucose (10.0 g)

Dextrose (5 g)

Nitrogen source

 

NaNO3 (15.0 g)

Urea (0.001 g)

Peptone (5.0 g), yeast extract (1 g)

Beef extract (10.0 g), yeast extract (5.0 g)

Minerals

K2HPO4 (0.5 g), FeSO4 (0.1 g)

FeSO4 (0.008 g), KH2PO4 (3.4 g), K2HPO4 (4.4 g), ZnSO4 (0.29 g/100 ml), CaCl2 (0.24 g), CuSO4 (0.25 g), MnSO4 (0.17 g)

KH2PO4 (1.0 g), FeCl2 (0.008 g)

Ferric citrate (0.1 g), sodium sulfate (3.24 g), calcium chloride (1.50 g), Na2HCO3 (0.16 g), H3BO3 (0.22 g), SnCl2 (0.034 g), sodium silicate (0.004 g), sodium fluorate (0.0024 g), NaNO3 (0.0016 g), Na2PO4 (0.008 g)

KH2PO4 (15.0 g), NH3SO4 (1.0 g), CaCl2 (0.02 g)

Other common ingredients

Na2CO3 (0.2 g), l-asparagine (0.1 g), MgSO4 (0.1 g)

KCl (1.1 g), MgSO4 (0.1 g), yeast extract (0.5 g)

MgSO4 (4.0 g), olive oil (10.0 g)

NaCl2 (19.45 g), MgCl2 (8.80 g), KCl2 (0.55 g), KBr (0.08 g)

Tryptose (10.0 g), casein (4.0 g), starch soluble (1.0 g), NaCl2 (5.0 g), MgSO4 (0.2 g)

Screening methods for potential producers

Hemolytic activity

Due to their amphiphilic character, surfactants induce hemolysis at a given concentration. Hemolytic activity in blood agar plate (peptone, 5 g; yeast extract, 3 g; NaCl2, 5 g; sheep blood, 5 ml) is a primary screening method to screen biosurfactant production [3, 9, 38]. Hemolytic activity was detected using blood agar plates with 5% (v/v) human blood. Plates were examined for hemolysis after incubation at 37°C for 24 h. The plates were inspected for zone of clearance around the colony. The presence of clearing zone served as an indicator of biosurfactant producing microorganism.

Drop collapsing test

Screening of biosurfactant production was performed using the qualitative drop-collapse test described by Youssef et al. [59]. In this method, mineral oil (2 μl) was added to 96-well microtitre plates. The plate was equilibrated for 1 h at 37°C and 5 μl of the culture supernatant was added to the surface of the oil. The shape of drop on the oil surface was observed after 1 min. The culture supernatant that makes the drop collapsed was indicated as positive result and the drops remain beaded were scored as negative, which are examined with distilled water as control.

Oil displacement test

Weathered crude oil (15 μl) was placed on the surface of 40 μl distilled water in a Petri dish and culture supernatant (10 μl) was gently put on the surface of the oil film. The diameter and area of clear halo visualized under visible light were measured after 30 s [39].

Screening for lipase production

The isolates that produce lipase were screened using tributyrin agar plates. Tributyrin (1%) was added to actinomycetes isolation agar. The pH of the medium was adjusted to 7.3–7.4 using 0.1 N NaOH. A loopful of inoculum was streaked on to the tributyrin agar plates. The plates were incubated at 26°C for 7 days. After incubation, the plates were examined for the formation of clear zone around the colonies.

Emulsification activity

Emulsification activity was performed according to Paraszkiewicz et al. [44]. Kerosene was added to CFS in a ratio of 1:1 and vortexed vigorously for 2 min. After 24 h of incubation, the height of the emulsified layer was measured. The emulsification index (E24) was estimated as
$$ E_{24} = H_{\text{EL}} /H_{\text{S}} \times 100 $$
where E24 is the emulsification activity after 24 h, HEL the height of the emulsified layer, and HS is the height of the total liquid column.

All the assays were performed in triplicate and compared with distilled water as control.

Identification of biosurfactant producer

Morphological and physiological characteristics of the strain were processed according to Lechavalier [30]. Colors of aerial and substrate mycelium were observed on different media including yeast extract–malt extract agar (ISP 2), oat meal agar (ISP 3), and inorganic salts starch medium (ISP 4). All the morphological characteristics of the strain were observed between 7 and 14 days of incubation. Microscopic morphology of the strain was observed with lactophenol cotton blue staining. Biochemical characteristics were determined with hydrolysis of gelatin, starch, cellulose, tributyrin and chitin. Freeze-dried cells were used to determine the chemotaxonomy of the cells to analyze the whole cell carbohydrate, amino acid and fatty acids of the strain N. alba MSA10.

The identification of biosurfactant producer was confirmed with phylogenetic analysis. Briefly, genomic DNA of N. alba MSA10 was extracted after Enticknap et al. [15]. Universal 16S rRNA eubacterial primer was used for the amplification of DNA. PCR amplified 16S rRNA gene product was cloned by the TA cloning method using a TOPO TA cloning® kit according to the manufacturer’s instructions (Invitrogen) for sequencing.

The 16S rRNA gene sequence obtained from the isolate Nocardiopsis MSA10 was compared with other bacterial sequences using NCBI BLASTn [1, 2] with a sequence query ‘biosurfactant’ for their pair-wise identities. Multiple alignments of these sequences were carried out by ClustalW 1.83 version of EBI (www.ebi.ac.uk/cgi-bin/clustalw/) with 0.5 transition weight. Phylogenetic trees were constructed in MEGA 4.0 version (www.megasoftware.net) using unweighted pair group method with arithmetic mean (UPGMA) algorithms. Transitions and transversion substitution matrixes and a heuristic search of 100 repetitions with random addition of sequences were performed. UPGMA bootstrapping was performed with 1,000 replicates with pseudo-random number generators. Nucleotide composition of each aligned sequence was predicted by BioEdit software package. The sequence used in the analysis was deposited in GenBank, EMBL in Europe and the DNA Data Bank of Japan with an accession number EU563352.

Production medium

The potential biosurfactant producer was cultured in different media (Table 1). The production medium (sodium propionate, 4.0 g; K2HPO4, 0.5 g; FeSO4, 0.1 g; Na2CO3, 0.2 g; l-asparagine, 0.1 g; MgSO4, 0.1 g; glucose, 1 g; peptone, 1 g; NaCl2, 1%; pH 7) was optimized in 1,000 ml Erlenmeyer flasks containing 600 ml of appropriate production medium. The inoculated flasks were incubated at 26°C for 7 days on a rotary shaker at 200 rpm.

Optimization of biosurfactant production

To find the optimum culture conditions for biosurfactant production, the production media were maintained at different temperatures (10, 20, 30, 40 and 50°C) and pH (4, 5, 6, 7, 8, and 9). Glucose, paddy straw, olive oil, kerosene, vegetable oil were supplemented to the production medium as carbon sources. Peptone, yeast extract, NaNO3, urea and beef extract were included in the optimization process as nitrogen sources. Salinity was optimized with different concentrations of NaCl2 ranged between 1 and 3.5%.

Extraction of biosurfactant

The strain N. alba was grown in a 1,000 ml Erlenmeyer flask containing 600 ml of actinomycetes isolation broth (Table 1) (Himedia®) and incubated at 26°C for 7 days at 200 rpm. The CFS was obtained by centrifugation at 8,000×g for 10 min at 4°C (Eppendorff 5804 R). The CFS was filtered through a 0.2 μm filter. Filtered supernatant and the harvested cells were used for the extraction of biosurfactant. Extraction was performed with liquid–liquid extraction and acid precipitation methods using CFS and harvested cells. CFS (500 ml) was collected and acidified with conc. HCl to attain pH 2.0 and extracted with an equal volume of solvents such as ethyl acetate, diethyl ether and dichloromethane. The resultant aliquot was concentrated to dryness in a rotary vacuum evaporator (Yamato DC 400) and the residue remained was tested for emulsification activity as described above.

Chemical analysis of biosurfactant

Analytical methods

The dried residue was analyzed for the quantification of macromolecules such as protein, carbohydrate, lipid and rhamnolipid. Protein was estimated using the method of Lowry et al. [31], carbohydrate was estimated by phenol–sulfuric acid method [11], lipid was estimated by the release of free fatty acids [49] and rhamnolipid was estimated using orcinol method [10].

Purification of biosurfactant

Solvent extract which showed the highest emulsification index was examined for purity using thin layer chromatography (TLC). Merck G silica gel plates (20 × 20 cm) were spotted with the evaporated residue and developed in different solvent system including 96% ethanol:water (7:3) for amino acids; chloroform:acetic acid:water (60:30:10) for sugars and chloroform:methanol:water (65:25:4) for lipids. Fractions were isolated and eluted with corresponding buffer and tested for emulsification activity to identify the active compound. The TLC fractions obtained were dissolved in 1 ml methanol and passed through a 0.22 μm pore filter. Active TLC fraction that confirmed emulsification was applied to liquid chromatography using semi-preparative HPLC (Shimadzu, Japan; HPLC RGM 011). The active filtrate was applied to HPLC on a reversed phase silica gel column. The column was eluted at a flow rate of 1.0 ml/min and observed at 254 nm. The concentration of biosurfactant was determined with peak calibration details of previous literatures. Eluted HPLC fractions were tested for emulsification activity and analyzed by TLC for the characterization of the compound.

Characterization of biosurfactant

Stability of the surface active compound was determined by dissolving the HPLC eluted fraction in 1–2% of NaCl and the emulsification index was calculated. The stability of the surface active compound at different pH values was performed by dissolving the biosurfactant in 0.1 M sodium acetate buffer (pH 4.0–7.0) and 0.1 M sodium phosphate buffer (pH 8.0–9.0). After 1 h of incubation with reciprocal agitation, the emulsification activity was measured as previously described. The stability of the surface active compound at different temperatures was carried out by incubating the surface active compound at 0.4% (w/v) in water for 30 min at 25–120°C before measuring the emulsification activity. All these factors were compared to that of the corresponding solution in water.

Antimicrobial activity of surface active compound

The HPLC-purified compound was tested for antimicrobial activity using well-diffusion method and area of the zone was calculated. Extracted active compounds were tested against human pathogens such as Candida albicans, E. coli, Proteus mirabilis, hemolytic Streptococcus, Pseudomonas aeruginosa, Micrococcus luteus, Staphylococcus epidermidis, Enterococcus faecalis, Klebsielle pneumoniae, Bacillus subtilis and Staphylococcus aureus. Mueller Hinton agar plates were prepared and swabbed with pathogens. Using cork borer, well was made and 50 μl of those extracted compound and the standard (10 μM amphotericin) was added in wells, incubated at 30°C for 24 h. After incubation, the clear zone was measured and calculated.

Results

Screening for biosurfactant production

In this study, all the four tests including hemolytic activity, oil displacement method, drop collapsing test and lipase activity used for the screening of biosurfactant producers showed MSA10 as a potent biosurfactant producer and was selected for further optimization processes.

Optimization of biosurfactant production

Growth curve and biosurfactant production in different culture conditions evidenced that maximum biomass and biosurfactant production started at 150 h of incubation, the onset of stationary phase. Biosurfactant production was increased to peak at early stationary phase (168 h). This may due to the release of cell bound biosurfactant into the culture broth which led to an increase in extra cellular biosurfactant production [13, 20]. Biosurfactant production of N. alba was optimized under different environmental and nutritional factors including temperature, pH, salinity, carbon sources, and nitrogen sources. Various media with different ingredients including mineral salt medium, actinomycetes isolation medium, actinomyces broth, ZoBell marine broth and the basal medium were used for the analysis of biosurfactant production (Table 1). The strain N. alba was able to grow and produce biosurfactant at all tested pH ranges, temperatures, and salt concentrations. The optimum biosurfactant production was observed at pH 8.0 (Fig. 1), temperature 30°C (Fig. 2), and 1% salt concentration (Fig. 3). Among the different media formulations, actinomycete isolation media showed highest yield (30%) of biosurfactant and biomass production (Fig. 4).
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Fig. 1

Effect of pH on biomass and production of biosurfactant by N. alba in AIA medium. Emulsification activity was determined in terms of emulsification index E24. Surfactant activity was measured using the clear zone diameter as determined by OST

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Fig. 2

Effect of temperature on biomass and production of biosurfactant by N. alba in AIA medium. Emulsification activity was determined in terms of emulsification index E24. Surfactant activity was measured using the clear zone diameter as determined by OST

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Fig. 3

Effect of salinity on biomass and production of biosurfactant by N. alba in AIA medium. Emulsification activity was determined in terms of emulsification index E24. Surfactant activity was measured using the clear zone diameter as determined by OST

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Fig. 4

Efficiency of biosurfactant and biomass production of N. alba was evaluated with different media including actinomycetes isolation agar, mineral salt medium, marine broth, actinomyces broth and basal medium. Various screening methods including hemolytic activity, oil displacement test, emulsification activity and lipase activity were carried out for the regimented production of biosurfactant

Influence of carbon sources

It has been envisaged that carbon sources have tremendous potential to support microbial growth and biosurfactant production [6, 14, 46]. The influence of carbon sources on the production of biosurfactant by N. alba was included in the optimization process. It was found that carbon sources induced the production of biosurfactant (Fig. 5). The supplementation of glucose and paddy straw to the culture medium increased biosurfactant production by 30 and 25%, respectively. The AIA medium normally contains 4 g sodium propionate as carbon source due to the combination of the ions in sodium propionate that reduces the production of biosurfactant but the supplementation of glucose in the medium increases the biosurfactant production. Based on the present findings, it was envisaged that the strain MSA10 might utilize the carbon sources as sole precursor for the production of secondary metabolites.
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Fig. 5

Effect of different carbon sources on biomass and production of biosurfactant by N. alba. Highest biosurfactant production was observed in paddy straw whereas the highest biomass production was effected in vegetable oil. Emulsification activity was measured using the emulsification index (E24) and surfactant activity was measured using the clear zone diameter as determined by OST

Influence of nitrogen sources

Supplementation of nitrogen sources to the production medium was not beneficial for the production of biosurfactant (Fig. 6). A greater repression of biosurfactant production was observed with beef extract, urea and NaNO3 supplemented to the production media. Whereas other nitrogen sources including peptone and yeast extract showed insignificant increase in the production of biosurfactant. Biomass production was high in peptone, beef extract and yeast extract and low in NaNO3 and urea. The supplementation of nitrogen sources resulted in increased biomass production, but not resulted in biosurfactant production. No correlation was established between biomass and biosurfactant production. Therefore, it could be inferred that the MSA10 can be used for biosurfactant production in industrial scale processes, since it does not require any nitrogen sources which may interfere with downstream processing.
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Fig. 6

Effect of different nitrogen sources on biomass and production of biosurfactant. Peptone and yeast extract showed highest biosurfactant production whereas the others do not show any production. Biomass production is also high in peptone and yeast extract the others showed low production

Extraction of biosurfactant

The CFS and pellet obtained were used for acid precipitation and tested for activity. The activity was determined by its presence in the CFS. Therefore, the CFS obtained with cold centrifugation was extracted with three different solvents including ethyl acetate, diethyl ether and dichloromethane. The solvent extractives were tested for hemolytic, oil displacement, drop collapsing, lipase and emulsification activity. The diethyl ether extract was found to have high emulsifying activity.

Comparison of synthetic surfactant with the biosurfactant

The emulsification activity of synthetic surfactant SDS was compared with the biosurfactant potential of N. alba. The emulsification activity of the diethyl ether extract showed similar activity (30%) as that of the synthetic surfactant SDS. The emulsifying activity of ethyl acetate and dichloromethane extractives was recorded 22.8 and 25%, respectively (Fig. 7).
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Fig. 7

Proficiency of biosurfactant production in N. alba was tested with different solvent extracts including ethyl acetate, diethyl ether, dichloromethane and compared with the synthetic surfactant SDS

Purification of biosurfactant

As reported previously, the diethyl ether extract which was obtained from the AIA medium was applied in TLC and HPLC for the characterization of the surface active compound. The TLC performed with diethyl ether extract gave a single spot with an Rf value of 0.77. In HPLC, elution was carried out at 25°C with 100% methanol at a flow rate of 1 ml/min and observed for the absorbance at 254 nm (Fig. 8). Purified compound from HPLC was applied in TLC and it gave the Rf value of 0.60. Chemical nature of the compound was similar to the structural characteristics of the lipopeptide compound. The compound constitutes substantial amount of carbohydrate 20 μg/0.1 ml, protein 35 μg/0.1 ml and lipid 573 μg/0.1 ml. The quantification of compound gave a higher ratio in lipid and protein compared to carbohydrate. Based on the analysis, the compound was partially confirmed as lipopeptide. The lipopeptide called surfactin, containing a carboxylic acid and seven amino acids, produced by the strain B. subtilis showed exceptionally high surface activity [21, 22]. Similarly, Hsieh et al. [24] reported that the concentration of lipopeptide compound from B. amyloliquifaciens S13 was 452.5 mg/l and B. subtilis ATCC21332 was 109.5 mg/l.
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Fig. 8

HPLC chromatogram showed the surfactant produced in spent growth medium by N. alba isolated from the marine sponge

Characterization of the biosurfactant

The HPLC-purified MSA10 biosurfactant fraction was analyzed under different environmental conditions including pH, temperature and salt concentrations to elucidate the stability of the surfactive compound. The biosurfactant activity showed stability between 10 and 80°C with only 10% loss of activity at 90°C. The compound was stable at a broad pH range between 4 and 9. The biosurfactant activity was restored up to 2% sodium chloride concentration.

Antimicrobial activity of biosurfactant

The purified biosurfactant compound showed highest antimicrobial activity against human pathogens (Fig. 9). The compound showed highest activity against E. faecalis and B. subtilis, it also showed activity against the pathogenic yeast C. albicans. However, it does not show activity against E. coli, hemolytic Streptococcus and P. aeruginosa.
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Fig. 9

Antimicrobial activity of the purified biosurfactant. Antimicrobial activity was tested against established human pathogens using well-diffusion method. The activity index was calculated as mm2 area based on the diameter halo produced by the biosurfactant

Characterization of MSA10

Based on the biochemical and physiological tests performed, the strain was identified as N. alba MSA10. This strain was a Gram positive non-motile rod and it was found to be catalase and methyl red negative. It utilized citrate and was found to be negative for Voges Proskauer test and positive for indole production. The 16S rRNA sequence of MSA10 was deposited in Genbank with an accession number EU563352. Taxonomic affiliation of the sequences was retrieved from classifier program of ribosomal database project (RDP-II) [55]. RDP-II hierarchy is based on the new phylogenetically consistent higher order bacterial taxonomy proposed by Garrity et al. [16]. The 16S rRNA sequence of the isolate MSA10 was further analyzed using NCBI BLASTn tool with a query to limit the search for closest biosurfactant producing relatives. Representatives of maximum homologous sequences from the search were used for the construction of phylogenetic tree using UPGMA algorithm. It was found that the isolate MSA10 showed clustering exclusively with biosurfactant producing Streptomyces strains. Closest major clusters were prominent biosurfactant producers: Bacillus and Pseudomonas (Fig. 10). These findings revealed that the isolate MSA10 was an Actinomycetes strain producing extracelluar biosurfactants. The neighboring cluster Bacillus and Pseudomonas genus are prominent lipopeptide producers and their functions include promotion of bacterial swarming [19] and biosurfactant properties [25]. In many cases, the lipopeptide compounds are known to exert a role in antagonistic interactions with other organisms [37, 40], including antifungal [25], antibacterial [18] and cytotoxic activity [21].
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Fig. 10

UPGMA bootstrapping phylogenetic tree of isolate MSI10 and their closest NCBI (BLASTn) biosurfactant producers based on the 16S rRNA gene sequences. Bootstrap values calculated from 1,000 resamplings using UPGMA are shown at the respective nodes when the calculated values were 50% or greater

Discussion

Sponges offer nourishment and a safe habitat to their symbionts; they hold more nutrient than seawater and sediments [7]. Marine microorganisms are good candidates for bioremediation, pharmaceutical, and bioactive natural products [28]. The strain N. alba chosen in this study was based on their potential surfactant production and its efficiency was judged with different screening methods mentioned above. Literature evidenced that nocardioform actinomycetes in general play a crucial role in the degradation of PAHs in soils [26].

Several conventional extraction methods used for the recovery of biosurfactants have been widely reported in literatures including acid precipitation, solvent extraction, crystallization, ammonium sulfate precipitation and centrifugation [12]. Generally, the solvents used for biosurfactant recovery such as acetone, methanol and chloroform are sometimes toxic in nature and harmful to the environment [32]. Cheap and less toxic solvents such as methyl tertiary butyl ether have been successfully used in recent years to recover biosurfactants produced by Rhodococcus [29]. These types of low cost, less toxic and readily available solvents can be used to reduce the recovery expenses substantially and minimize environmental hazards. The present findings agree with the report of Kuyukina [29] in which the solvent diethyl ether showed highest recovery of biosurfactant.

Many studies showed glucose as the best carbon source for the production of lipopeptide biosurfactant [42, 44]. While analyzing the carbon sources, the cheapest raw material, paddy straw, showed highest production of biosurfactant. According to Ouled Hadder et al. [43], NaNO3 promotes good growth but it does not enhance the productivity. In our study, NaNO3 promotes the growth with less emulsification activity. Other nitrogen sources including peptone and yeast extract increase the growth and emulsification activity.

Cyclic lipopeptides are produced by distinctively different groups of bacteria, both Gram positive [27] and Gram negative [37]. The high diversity of lipopeptide producing microorganisms and difference in chemical structure suggest that the lipopeptide compounds may serve different, and possibly multiple, purposes. Due to the limited applications of lipopeptides, only few works have been reported including bacterial swarming and biosurfactant properties [19, 26]. Several lipopeptide producing organisms can hold potent antimicrobial property [34, 35, 54, 58].

Due to its advantages of low toxicity [45], biodegradability [60], environmental compatibility [17], better foaming properties and stable activities at extremes pH, salinity and temperature [12], biosurfactants can be used in various applications. Few recent findings have been reported in the extracellular biosurfactant production from marine environment. For example, the marine bacterium Myroides sp. [33] produces biosurfactant in the culture supernatant. The present findings elucidated the highest yield of biosurfactant extracellularly by the strain N. alba MSA10.

It is possible to protect the marine environment with the use of biosurfactants because a number of marine bacterial strains can produce biosurfactant during growth on hydrocarbons [5, 41]. Based on the present findings and general trend observed with other sponge-associated actinomycetes, it has been hypothesized that the production of biosurfactants by the sponge-associated bacteria might have a significant role in the chemical ecology of host sponge. However, the hypothesis has not been tested in controlled in vivo experiments. Considering the established functions of biosurfactants including anti-adhesive/biofilm disruption activity [23, 36], the biosurfactants may be involved in host defense against fouling processes. Our research group has recently established the sponge-associated bacteria as an indicator of heavy metal pollution in marine environment [51]. The functional role of biosurfactant production by the sponge-associated bacteria in marine environment is under investigation.

Conclusion

Literature evidenced that marine actinomycetes are untapped resource for biosurfactant production. Considering the need of new strains for the production of novel surfactive molecules, the present study brings out a new insight on the exploration of marine environment for biosurfactant production and process optimization for industrial applications. Biosurfactants have been proving as the promising agents for bioremediation of hydrocarbons, heavy metals and oil-polluted marine environments. Therefore, explorations of marine environment for the biosurfactant producers will have wider applications in industrial processes, bioremediation and exploring the chemical ecology of sedentary marine organisms.

Acknowledgments

RG is thankful to CSIR, New Delhi for the award of Senior Research Fellowship in CSIR funded research project. This paper is an outcome of CSIR project No. 38(1128)/06/EMR-II.

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© Springer-Verlag 2009