Production and characterization of lipopeptide biosurfactant by a sponge-associated marine actinomycetes Nocardiopsis alba MSA10
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- Gandhimathi, R., Seghal Kiran, G., Hema, T.A. et al. Bioprocess Biosyst Eng (2009) 32: 825. doi:10.1007/s00449-009-0309-x
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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.
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 . Bacteria constitute up to 60% of the biomass of sponges . If some compounds are derived from symbiotic microorganisms, it could provide an improved sustainable source of supply for bioactive compound . 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 . 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 . According to Rosenberg and Ron , 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 . 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
Composition of culture media used for biosurfactant production by N. alba MSA10
Nature of ingredients
Sodium propionate (4.0 g)
Glucose (10.0 g)
Glucose (10.0 g)
Glucose (10.0 g)
Dextrose (5 g)
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)
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
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. . 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 .
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.
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 . 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. . 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.
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
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. , carbohydrate was estimated by phenol–sulfuric acid method , lipid was estimated by the release of free fatty acids  and rhamnolipid was estimated using orcinol method .
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.
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
Influence of carbon sources
Influence of nitrogen sources
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
Purification of biosurfactant
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
Characterization of MSA10
Sponges offer nourishment and a safe habitat to their symbionts; they hold more nutrient than seawater and sediments . Marine microorganisms are good candidates for bioremediation, pharmaceutical, and bioactive natural products . 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 .
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 . Generally, the solvents used for biosurfactant recovery such as acetone, methanol and chloroform are sometimes toxic in nature and harmful to the environment . Cheap and less toxic solvents such as methyl tertiary butyl ether have been successfully used in recent years to recover biosurfactants produced by Rhodococcus . 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  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. , 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  and Gram negative . 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 , biodegradability , environmental compatibility , better foaming properties and stable activities at extremes pH, salinity and temperature , 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.  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 . The functional role of biosurfactant production by the sponge-associated bacteria in marine environment is under investigation.
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.
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.