Production of a new glycolipid biosurfactant from marine Nocardiopsis lucentensis MSA04 in solid-state cultivation

Colloids and Surfaces B: Biointerfaces 78 (2010) 8–16

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Production of a new glycolipid biosurfactant from marine Nocardiopsis lucentensis MSA04 in solid-state cultivation G. Seghal Kiran, T. Anto Thomas, Joseph Selvin ∗ School of Life Sciences, Bharathidasan University, Tiruchirappalli 620 024, India

a r t i c l e

i n f o

Article history: Received 18 September 2009 Received in revised form 14 January 2010 Accepted 28 January 2010 Available online 4 February 2010 Keywords: Biosurfactant Marine actinobacterium Optimization Response surface methodology Glycolipid biosurfactant

a b s t r a c t Considering the need of potential biosurfactant producers and economic production processes using industrial waste, the present study aims to develop solid-state culture (SSC) of a marine actinobacterium for biosurfactant production. A potential biosurfactant producer Nocardiopsis lucentensis MSA04 was isolated from the marine sponge Dendrilla nigra. Among the substrates screened, wheat bran increased the production significantly (E24 25%) followed by oil seed cake and industrial waste such as tannery pretreated sludge, treated molasses (distillery waste) and pretreated molasses. Enhanced biosurfactant production was achieved under SSC conditions using kerosene as carbon source, beef extract as nitrogen source and wheat bran as substrate. The maximum production of biosurfactant by MSA04 occurred at a C/N ratio of 0.5 envisaging that a higher amount of nitrogen source is required by the strain compared to that of the carbon source. The kerosene and beef extract interactively increase the production and a stable production was attained with the influence of both factors independently. A significant interactive influence of secondary control factors such as copper sulfate and inoculum size was validated in response surface methods-based experiments. The surface active compound produced by MSA04 was characterized as glycolipid with a hydrophobic non-polar hydrocarbon chain (nonanoic acid methyl ester) and hydrophilic sugar, 3-acetyl 2,5 dimethyl furan. In conclusion, the strain N. lucentensis MSA04 was a potential source of glycolipid biosurfactant, could be used for the development of bioremediation processes in the marine environment. © 2010 Elsevier B.V. All rights reserved.

1. Introduction Biosurfactants are heterogeneous group of secondary metabolites with surface active properties and are synthesized by a variety of microorganisms [1]. Busnell and Haas [2] were among the first to demonstrate bacterial production of biosurfactants. They are amphiphilic in nature and contain atleast one hydrophilic and hydrophobic moiety [3]. These amphiphilic compounds are produced on living surfaces or excreted extracellularly and also had the ability to accumulate between fluid phases, thus reducing surface tension and interfacial tension at the surface and interface, respectively [4]. According to Desai and Banat [5], biosurfactants constitute glycolipids, phospholipids, lipoproteins or lipopeptides, polymeric compounds, mycolic acids and lipopolysaccharides. Biosurfactants are attracting attention in recent years because they offer several advantages over chemical surfactants due to its low

∗ Corresponding author at: Department of Microbiology, Bharathidasan University, Palkalaiperur, Tiruchirappalli 620024, Tamil Nadu, India. Tel.: +91 431 2407082; fax: +91 431 2407045. E-mail addresses: [email protected], [email protected] (J. Selvin). 0927-7765/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2010.01.028

toxicity, inherent good biodegradability and ecological acceptability. Production economy is the major setback in biosurfactant production, as the amount and type of raw materials can contribute considerably to the production cost. It is estimated that the raw materials account for 10–30% of the total production cost in most bioprocesses. Thus to reduce this cost it is desirable to use lowcost raw materials for the production of biosurfactants [6]. One possible strategy for reducing costs is the utilization of alternative substrates such as agro-industrial wastes [7]. A variety of cheap raw materials, including plant-derived oils, oil wastes, starchy substances, lactic whey and distillery wastes have been reported to support biosurfactant production. Agro-industrial wastes including oil wastes from vegetable-oil refineries and the food industry were also reported as good substrates for biosurfactant production [8,9]. Recently, statistical experimental strategies including factorial design and response surface methodology (RSM) have been successfully employed for the optimization of solid-state culture [10–13]. These strategies are particularly suited for the optimization of solid-state culture (SSC) because they are generally subjective to less accidental errors. Considering the need of potential biosurfactant producers and economic production pro-

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cesses using industrial waste, the present study aims to explore the marine actinobacteria, an unexplored resource for the production of biosurfactants using industrial and agro-industrial wastes. In the present study, the production process was developed under solid-state culture (SSC), an economic process scarcely used for the production of biosurfactants from actinobacteria. 2. Materials and methods 2.1. Isolation and culture of sponge-associated marine actinobacteria Marine sponge Dendrilla nigra was collected from the 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 for kept 2 h in sterilized aged seawater (SAS) to remove loosely associated microorganisms from inner and outer sponge surfaces. It has been hypothesized that this process may eliminate non-associated 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 sponge-associated actinobacteria, 1 cm3 of sponge tissue was excised from the internal mesohyl area using a pair of sterile scissors. The excised portion was thoroughly washed three times with sterile SAS 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 SAS and preincubated at 40 ◦ C for 1 h for the activation of dormant cells. The aliquot was placed on various isolation media including marine sponge agar and standard media (HiMedia) [14]. 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 (HiMedia) at 4 ◦ C. 2.2. Screening for biosurfactant producers Biosurfactant production was examined with drop collapsing [15] and oil displacement tests [16]. Hemolytic activity was performed on blood agar plates containing 5% (v/v) human blood. For screening the isolated strains, 50 ␮l broth culture of the isolates was spot inoculated on to blood agar plates and incubated at 37 ◦ C for 24 h. The plates were visually inspected for zone of hemolysis around the colony. The diameter of the zone of hemolysis is a qualitative method used as an indicator of biosurfactant production. Lipase activity was measured using the tributyrin agar plates. Chromogenic substrate plates were prepared by using phenol red (0.01%) along with 1% lipidic substrate [tributyrin (Himedia)/triolein (Sigma)/olive oil/corn oil], 10 mM CaCl2 , and 2% agar. The pH was adjusted to 7.3–7.4 by using 0.1N NaOH. A loopful of inoculum was streaked on the tributyrin agar plates and a clear zone around the inoculum indicated the production of lipase. Emulsification activity was performed according to Paraszkiewicz et al. [17]. Kerosene was added to cell free broth in a ratio of 1:1 and vortexed vigorously for 2 min. All the assays were performed in triplicate with distilled water as the negative control. After 24 h of incubation, the height of the emulsified layer was measured and the emulsification index (E24 ) was estimated as follows: E24 =

HEL × 100 HS

E24 is the emulsification activity after 24 h, HEL is the height of the emulsified layer and HS is the height of the total liquid column.

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2.3. Identification of biosurfactant producer The strain MSA04 was identified morphologically and biochemically according to the method of Lechavalier and Lechavalier [18] and the genomic DNA was obtained by the method of Ferrara et al. [19]. For the 16S rRNA sequencing the PCR analysis was performed as follows: universal 16S rRNA eubacterial primer (5 -GAGTTTGATCCTGGCTCAG-3 ; 5 -AGAAAGGAGGTGATCCAGCC3 ) was used for the amplification of DNA. The polymerase chain reaction was carried out on a thermal cycler (Eppendorf) in a 50 ␮l reaction mix. The reaction mix contained 10× amplification buffer (5 ␮l), 1.5 mM MgCl2 (5 ␮l), 1 ␮l forward primer (10 mM), 1 ␮l reverse primer (10 mM), 1 ␮l dNTP, and 0.25 ␮l Taq polymerase. After initial denaturation at 95 ◦ C for 1 min, amplification was performed with 35 cycles of 35 s at 94 ◦ C, 40 s at 55 ◦ C, 2 min at 72 ◦ C followed by a final extension at 72 ◦ C for 8 min. The PCR products were visualized by electrophoresis through a 1.2% agarose gel (Genei). The 16S rRNA gene sequence obtained from the isolate MSA04 was compared with other bacterial sequences by using NCBI megaBLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) for their pair wise identities. Multiple alignments of these sequences were carried out by Clustal W 1.83 version of EBI (www.ebi.ac.uk/cgibin/clustalw/) with 0.5 transition weight. Phylogenetic trees were constructed in MEGA 4.0 version (www.megasoftware.net) using neighbor joining (NJ), minimum evolution (ME) and unweighted pair group method with arithmetic mean (UPGMA) algorithms. Nucleotide composition of each aligned sequence was predicted by BioEdit software package and the 16S rRNA sequence of MSA04 was deposited in NCBI with an accession number GQ153942. 2.4. Optimization of biosurfactant production under solid-state culture (SSC) For the development of SSC, the production substrate was developed using agro-industrial and industrial waste. To find suitable substrate for the development of SSC, agro-industrial waste such as wheat bran, ground nut oil cake, rice bran, and oil seed cake and industrial waste such as furnished leather powder, diesel contaminated soil, petrol bunk soil, AavinTM (milk processing waste) pretreated sludge, AavinTM (milk processing waste) treated waste, tannery pretreated sludge, treated molasses (distillery waste), pretreated molasses (distillery waste), and tannery treated sludge were used for the screening. Based on the screening results, six substrates including the industrial and agro-industrial wastes such as oil seed cake, wheat bran, tannery treated sludge, tannery pretreated sludge, treated molasses and pretreated molasses were used in the SSC development. 2.4.1. Production of biosurfactant under SSC In the present study, new SSC media was developed for the optimization of biosurfactant production. The production of biosurfactant was performed in triplicate 250 ml Erlenmeyer flasks. To develop the SSC, 5 g of the dried substrate was transferred to 250 ml Erlenmeyer flasks and mixed with 6 ml of moistening media (A) (pH 7.0) and 7 ml of sterile distilled water. The composition of the salt solution was NH4 NO3 0.5%, NaCl 0.9%, MgSO4 ·7H2 O 0.1% and pH 7. The contents were double sterilized by autoclaving at 103.4 kPa for 20 min. The sterilized solid substrate was inoculated with 2.5 ml of the spore inoculum. The contents were mixed properly and incubated at 30 ◦ C for 7 days. 2.4.2. Efficacy of solvents on the extraction of surface active compounds The fermented substrate was mixed well with 50 ml of distilled water using a magnetic stirrer (Remi). The mixture was filtered

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through a filter paper, centrifuged and checked for emulsification activity. The collected extract was adjusted to pH 2 with 1N HCl. Equal volume of solvents either of acetone, dichloromethane, ethyl acetate or chloroform:methanol of 2:1 ratio and water was added to the acidified supernatant and stored over night in a refrigerator. The organic phase of the mixture was evaporated using a rotary vacuum evaporator (Yamato) and the concentrated extract evaluated for emulsification activity.

2.4.3. Optimization of biosurfactant production Optimization of biosurfactant production was carried out by search one at a time technique. Subsequently appropriate experimental models were developed in order to study the interactions between the factors. Factors like carbon and nitrogen sources, pH, temperature and salt concentration affecting the secretion of biosurfactant production were determined. The factors used for the biosurfactant production are presented in Table 1. The optimization process was completed with the effective concentration of chosen carbon and nitrogen source to maximize the production under SSC conditions. Most appropriate carbon and nitrogen sources were selected from the range of these sources used in the optimization. The SSC was conducted with 1%, 2%, 3% and 4% of the respective carbon and nitrogen sources. The glucosamine based biomass estimation [20] was used to quantify the biomass, since direct estimation of biomass is not possible in SSC.

2.5. Optimization of biosurfactant production using response surface methodology RSM [21,22] is an empirical technique employed for multiple regression analysis by using quantitative data obtained from properly designed experiments to solve multivalent equations simultaneously. The RSM was a novel approach used to analyse the experimental design data. Box–Behnken design (BBD) is a fractional factorial design obtained by combining two-level factorial designs with incomplete block designs. In order to be correlated to the independent variables, the response variable was fitted by a second order model. The statistical software package, Design-Expert 7.0 (Stat-Ease, Inc., Minneapolis, MN, USA) was used for the regression analysis of the experimental data, and also to plot the response surface graphs. The statistical significance of the model equation and the model terms was evaluated with Fisher’s test. In the present study, the critical control factors influenced the biosurfactant production by MSA04 was used in the RSM-based experiments. In the experimental design, four variables (kerosene, beef extract, copper sulfate and inoculum size) that have effect on the production of surfactant were identified by optimization experiments. Each independent variable was investigated at a high (+1), middle (0), and a low (−1) level. Runs of center points (control) were included in the matrix.

Table 1 Factors considered for the optimization of biosurfactant production in SSC. Factors

Ranges

pH Temperature Salinity Carbon sources Nitrogen source Metals Aminoacids Inoculum size Incubation time

5–9 (with increments of 1) 10–50 ◦ C (with increments of 10 ◦ C) 0.5–5% (with increments of 0.5%) Glucose, olive oil, kerosene, vegetable oil (1%) Yeast extract, beef extract, (NH4 )2 NO3 , acrylamide FeSO4 , CuSO4 , MnCl2 , MgCl2 (1%) Asparagine, valine, leucine, glycine, glutamic acid (1%) 1.0, 1.5, 2.0, 2.5, 3.0 ml 5–8 days

2.6. Characterization of surface active compound 2.6.1. Biochemical characterization The macromolecule present in the surface active extract was estimated for protein, lipid and carbohydrate. The protein was estimated using the method of Lowry et al. [23], carbohydrate and lipid by the methods of Chaplin and Kennedy [24] and Sadasivam and Manickam [25], respectively. The glycolipid concentration is based on the method of Chandrasekaran and Bemiller [1]. 2.7. Purification and chemical analysis of the surface active compound To purify the surface active compound, the concentrated extract was subjected to column chromatography on reverse phase silica gel (230–400 mesh) with step wise elution using methanol from 65% to 100% at a flow rate of 0.5 ml/min at room temperature. The active fraction was confirmed by the emulsification activity and the purity was checked by TLC. TLC was performed for proteins (n-butanol:acetic acid:water 4:3:2), carbohydrates (chloroform:aceticacid:water 60:30:10) and lipids (chloroform:methanol:water 65:25:4). The crude extract was fractionated using a silica gel column (30 cm × 2.0 cm) and eluted with the solvent system (MeOH:H2 O) prepared in increasing polarities. Fractions of 12 ml each were collected, and used for reading the optical density at 220 nm using a UV–vis Spectrophotometer (PG Instruments, UK). The GC–MS (a 5890II Hewlett-Packard gas chromatograph coupled to a HP5972A mass spectrophotometer integrated with HP MS Chemstation) and 1 H nuclear magnetic resonance (NMR) analysis were performed using the active fraction to elucidate the chemical structure of the biosurfactant. 2.7.1. Stability of the purified surface active compound Stability of the surface active compound was determined under various ranges of NaCl, pH and temperatures. The HPLC eluted active fraction in 1–5% of NaCl was evaluated by determining the emulsification index. The stability of the surface active compound at different pH values were 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 from 25 to 120 ◦ C before measuring the emulsification activity. All these factors were compared to that of the corresponding solution in water. 2.8. Determination of biosurfactant concentration and critical micelle concentration Biosurfactant concentration was determined by a gravimetric method described by Nitchke and Pastore [26]. The extract collected after centrifugation was acidified to pH 2.0 using 1N HCl and kept for overnight at 4 ◦ C, and then centrifuged at 10,000 × g for 20 min. The resultant supernatant was discarded and the remaining pellet was dried at 37 ◦ C till constant weight was obtained. The net weight of the crude precipitate was determined, and the crude surfactant concentration (in g/l) was calculated. To extrapolate the surfactant activity of purified compound with emulsification index, the surface tension was measured with a tensiometer using the duNouy procedure (Sigma) with a platinum ring at 24 ◦ C. The surface tension–concentration plots were used to determine critical micelle concentrations. In the present study, SDS was used as standard.

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2.9. Hydrocarbon degradation Hydrocarbon degradation potential of biosurfactant produced by the strain MSA04 was evaluated in vitro using the cell surface hydrophobicity assay [27] and cell aggregation assay [28]. The bacterial adhesion to hydrocarbon (BATH) method for the determination of bacterial cell surface hydrophobicity was used to evaluate the biodegradation potential of the strain MSA04. Experiments were carried out for 7 days using the procedure described by Rosenberg et al. [29]. Microbial hydrophobicity was measured in exponential growth phase. Buffer PUM (g l−1 ): 19.7 K2 HPO4 , 7.26 KH2 PO4 , 1.8 urea and 0.2 MgSO4 ·7H2 O was used for the hydrophobicity test. After centrifugation, cells were washed twice with PUM buffer, suspended in the PUM buffer to fit an optical density of ca. 1.0 (A0 ). Optical density was measured at 600 nm on a UV–vis Spectrophotometer (PG Instruments, UK). Then, 500 ␮l of hydrocarbon was added to 5 ml of microbial suspension and vortexed for 2 min. After 10 min, the optical density of the aqueous phase was measured (A1 ). The degree of hydrophobicity is calculated as [1 − (A1 /A0 )] × 100 [%]. 2.10. Search for biosynthetic genes Biosynthetic genes including polyketide synthases, nonribosomal peptide synthases, and genes encode for rhamnolipid and lipopeptide biosynthesis were targeted to elucidate possible mechanism of biosynthesis of surface active compound. Specific sets of degenerate primers [30–32] targeting the genes encoding polyketide synthases and non-ribosomal peptide synthetase (NRPS) were used for amplification in a touchdown PCR with a DNA as template. The resultant PCR products were purified and cloned using TOPO TA cloning kit (Invitrogen) for sequencing. The rhamnosyl transferase (rhlB) gene is a part of rhamnolipid biosynthesis pathway. The forward and reverse primers (5 -GCCCACGACCAGTTCGAC-3 ; 5 CATCCCCCTCCCTATGAC-3 ) homologous to 1378 bp of rhlB gene was designed (http://www.yeastgenome.org/) and used for the amplification of MSA19 rhlB gene. The kpd1/kpd2 primer set [33] was used to confirm the rhlB gene amplicon. The sfp 675 bp fragment [34], corresponding to the sfp gene (GenBank accession no. X63158) at positions 167–841, was PCR amplified using two oligonucleotide primers of sfp-f (5 -ATG AAG ATT TAC GGA ATT TA-3 ) and sfp-r (5 -TTA TAA AAG CTC TTC GTA CG-3 ). 3. Results 3.1. Screening for potential biosurfactant producers Based on the stability in subculturing, only 57 isolates (MSA01–MSA57) were retained and used for the screening of potential biosurfactant producers. Biosurfactant producers were screened using the cell free supernatant. Based on the screening tests performed, the 17 isolates were screened as biosurfactant producers. Among these, five isolates were grouped as potential biosurfactant producers. In the present study, we report the biosurfacant production potential of MSA04. The results of screening tests performed on MSA04 include hemolytic activity (8 mm), oil displacement (8 mm), lipase activity (80 U/mg), positive drop collapsing test and emulsification activity (15%). 3.2. Characterization and identification of biosurfactant producer MSA04 The strain MSA04 was non-motile, Gram positive and showed unique spores in Ziehl Nielsen staining. The isolate MSA04 was MRVP positive, utilized glucose and positive in the hydrolysis of starch,

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gelatin, cellulose and tributyrin. The isolate not utilized mannitol. The isolate was sensitive to ciprofloxacin and chloramphenicol but resistant to ampicillin and tetracycline. Taxonomic affiliation of the isolate was retrieved from classifier programme of Ribosomal Database Project II (RDPII) (http://rdp.cme.msu.edu). Representative of maximum homologous (97–99%) sequences of each isolate were obtained from seqmatch programme of RDPII and were used for the construction of phylogenetic affiliation. The isolate MSA04 showed unique branching from other nearest Nocardiopsis clusters (Fig. 1). Based on the morphological, biochemical characteristics, results of RDPII SEQMATCH programme and phylogenetic analysis, the isolate was identified as Nocardiopsis lucentensis MSA04. 3.3. Biosurfactant production and its culture conditions In the preliminary experiments, biosurfactant production by N. lucentensis MSA04 was performed in submerged culture. But the production was not increased significantly (data not shown) and therefore the optimization was performed in SSC. Among the substrates screened for the production of biosurfactant by N. lucentensis, wheat bran increased the production significantly (E24 25%) followed by 20% production in the oil seed cake and industrial waste such as tannery pretreated sludge, tannery treated sludge and treated molasses (distillery waste). The influence of carbon sources such as glucose, olive oil, vegetable oil and kerosene was evaluated under SSC conditions for the enhanced production of biosurfactant by N. lucentensis. The strain N. lucentensis resulted in enhanced biosurfactant production under SSC using kerosene as carbon source. The kerosene invariably increased the biosurfactant production with various substrates such as wheat bran (68%), oil seed cake (58%), tannery treated sludge (55%), tannery pretreated sludge (55%), pretreated molasses (52%) and treated molasses (48%). Kerosene was found to be the best carbon source for the production of biosurfactant when compared to other carbon sources like glucose, olive oil and vegetable oil. Supplementation of nitrogen sources in the production media showed substantial increase in the biosurfactant production by N. lucentensis MSA04. However the effect of nitrogen sources on the biosurfactant production was greatly influenced by substrates under SSC conditions. In the present study, the nitrogen sources such as beef extract, yeast extract, sodium nitrate, ammonium nitrate and acrylamide were evaluated for the enhanced biosurfactant production under SSC using various substrates including oil seed cake, wheat bran, tannery treated sludge, tannery pretreated sludge, treated molasses and pretreated molasses. The beef extract as nitrogen source and wheat bran as substrate showed significant increase in the production of biosurfactant (60%) by N. lucentensis MSA04. The moisture content required for the maximum production of biosurfactant by N. lucentensis MSA04 under SSC was determined to be >80% and maximum production occurred in the substrate inoculated with 2.5 ml of inoculum. The inoculum was developed after 5 days of subculture in actinomycetes broth. The production was consistent at pH 7.0 on all the substrates used. The production was drastically declined at lower pH when compared to the higher pH 9.0, where the production was nearly stable. The enhanced biosurfactant production (55–70%) by N. lucentensis MSA04 was attained at pH 7.0. The production of biosurfactant was greatly affected by the fluctuation of incubation temperature. The production of biosurfactant by N. lucentensis MSA04 reached maximum at 25 ◦ C with wheat bran as substrate. Maximum biosurfactant production occurred on the supplementation of 2% NaCl. The production was almost stable in NaCl concentrations between 0.5% and 5%. The influence of metal ions such as CuSO4 , MnCl2 , MgSO4 and FeCl3 on the production of biosurfactant by N. lucentensis MSA04 was evaluated under SSC

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Fig. 1. NJ bootstrapping phylogenetic tree of isolate MSA04 and their closest NCBI (BLASTn) biosurfactant producers based on the 16S rRNA gene sequences. Bootstrap values calculated from 1000 resamplings using UPGMA are shown at the respective nodes when the calculated values were 50% or greater.

conditions. Among the metal ions screened, CuSO4 was shown to be the most appropriate metal ion for the production of biosurfactant by N. lucentensis MSA04 followed by FeCl3 . An enhanced biosurfactant production by N. lucentensis MSA04 was achieved with CuSO4 with wheat bran as substrate. The strain utilizes the amino acid, asparagine than other amino acid like glycine, leucine and valine for maximum biosurfactant production. The maximum biosurfactant production occurred on 7th day of incubation with wheat bran as substrate. The glucosamine based biomass estimation indicated that the biomass invariably reached the exponential phase on the 3rd day of growth and the stationary phase started on the 7th day of incubation. Albeit the relationship between biomass and biosurfactant production was not established in the present study, the production was invariably started at the late exponential phase and almost consistent up to stationary phase. In the present study, the optimization process was completed with the effective concentration of chosen carbon and nitrogen source to maximize the production under SSC conditions. Most appropriate carbon and nitrogen sources were selected from the range of these sources used in the optimization. The SSC was conducted with 1%, 2%, 3% and 4% of the respective carbon and nitrogen sources. The strain N. lucentensis MSA04 produced maximum biosurfactant with kerosene as carbon source and yeast extract as nitrogen source. The concentration of these sources and substrates used influenced the production significantly. The strain MSA04 produced maximum biosurfactant using the SSC with 1% kerosene as

carbon source (Fig. 2) and wheat bran as substrate. The production was consistently increased on the treated molasses supplemented with 2% beef extract as nitrogen source (Fig. 3). The maximum production of biosurfactant by MSA04 occurred at a C/N ratio of 0.5 envisaging that a higher amount of nitrogen source is required by the organism compared to that of the carbon source.

Fig. 2. Effect of increasing concentration of optimized carbon source, kerosene on the production of biosurfactant by N. lucentensis MSA04. The substrates used were oil seed cake (OSC), wheat bran (WB), tannery treated sludge (TT), tannery pretreated sludge (TPT), treated molasses (TM) and pre-treated molasses (PTM).

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Fig. 4. Contour plot of the interaction between the kerosene (carbon source) and beef extract (nitrogen source) on the production of biosurfactant in SSC by N. lucentensis MSA04.

Fig. 3. Effect of increasing concentration of optimized nitrogen source, beef extract on the production of biosurfactant by N. lucentensis MSA04. The substrates used were oil seed cake (OSC), wheat bran (WB), tannery treated sludge (TT), tannery pretreated sludge (TPT), treated molasses (TM) and pre-treated molasses (PTM).

3.4. Optimization under RSM-based experimental design The R2 value of 0.8087 which is closer to 1 showed the model to be stronger and it can better predict the response. The model was found to be significant with p ≤ 0.0156 (Table 2). The behavior of the system was explained by the following quadratic model equation: Y = 64.67 − 5.08X1 − 3.5X2 + 0.92X3 + 1.17X4 + 10.92X12 − 19.96X22 − 20.08X32 − 3.46X42 + 2.75X1 X2 + 4X1 X3

in SSC and kerosene, beef extract, CuSO4 and inoculum size are the critical control factors. 3.5. Extraction of surface active compound Among the various solvent systems including acetone, dichloromethane, ethyl acetate, water and chloroform:methanol (2:1) used for the extraction of surface active compounds, the chloroform:methanol (2:1) was found to be highly effective solvent system. The surface active compound was completely soluble in methanol, chloroform, and Tris-HCl buffer not in any other solvent system tested like ethyl acetate, DMSO, hexane, acetone and dichloromethane. 3.6. Purification and chemical analysis

+ 2.5X1 X4 − 4X2 X3 − 0.75X2 X4 − 11.25X3 X4 The predicted value of Y by the above-explained quadratic model was found to be 73%. The experimental value obtained was 76%. In the present study, the carbon and nitrogen sources were considered as primary critical control factors and the remaining factors were considered as secondary control factors. It was found that the production was significantly influenced by the variables such as kerosene, beef extract, copper sulfate and inoculum size either interactively and/or independently. The kerosene and beef extract interactively increase the production and the stable production was attained with the influence of both factors independently (Fig. 4). A significant interactive influence of secondary control factors such as copper sulfate and inoculum size was validated in the RSM design. The copper sulfate and inoculum size interactively reached a central value to influence the production maxima over a stable area (Fig. 5). In conclusion, the biosurfactant production by MSA04 was increased to 2.5-fold over the wild strain. For the production of biosurfactant by MSA04, wheat bran was the highly effective substrate

The surface active extract of MSA04 contained 0.123 mg/ml protein, 0.972 mg/ml carbohydrate, 1.886 mg/ml lipid and 9.0 mg/ml glycolipid. Based on the TLC analysis, carbohydrate and lipid with an Rf value of 0.8 were separated. The GC–MS analysis showed that the compound produced by MSA04 was a glycolipid with a hydrophobic non-polar hydrocarbon chain (nonanoic acid methyl ester) (Fig. 6) and hydrophilic part of the sugar compound was identified as 3-acetyl 2,5 dimethyl furan (Fig. 7). Retention times, relative intensities (%) and EIMS of the relevant peaks are as follows—peak A: 8.594, 40, EIMS m/z (% rel. intensity), 140 (45), 120 (100), 80

Table 2 ANOVA analysis of the optimization of production by N. lucentensis MSA04. Factors

Terms

p-Value

Kerosene Beef extract Copper sulfate and inoculum size Kerosene Beef extract Copper sulfate

Linear Linear Interaction Squared Squared Squared

0.1657* 0.3295* 0.0837* 0.0562* 0.0023*** 0.0022***

* ***

Affecting terms. More significant.

Fig. 5. Contour plot of the interaction between the inoculum size and CuSO4 on the production of biosurfactant in SSC by N. lucentensis MSA04.

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Fig. 6. The mass spectrum of hydrophilic part (3-acetyl 2,5 dimethyl furan) MSA04.

Table 3 Predicted structural moieties of biosurfactants produced by N. lucentensis MSA04. Strain

MSA4

Fig. 8. Hydrophobicity of surfactive compound produced by N. lucentensis MSA04. () Hydrophobicity (BATH method) in the presence of emulsified kerosene; () hydrophobicity in the presence of emulsified diesel; () hydrophobicity in the presence of emulsified hexane; (×) hydrophobicity of actinobacterial cells during growth in the presence of biosurfactive compound at different concentrations.

Compound Hydrophobic moiety

Hydrophilic moiety

Nonanoic acid methyl ester

3-Acetyl 2,5 dimethyl furan

factor for the production of biosurfactant. The purified biosurfactant from the strain MSA04 was stable over NaCl concentrations (1–4%). The emulsification index of biosurfactant produced by N. lucentensis MSA04 was invariably high over the synthetic surfactants such as SDS, tween20 and tween80. It was found that the emulsion formed was stable for more than 3 months. 3.8. Hydrocarbon degradation

(30), 50 (18), 40 (100), 15 (15). Peak B: 10.625, 60, EIMS m/z (% rel. intensity), 186 (5), 143 (5), 127 (10), 100 (10), 87 (40), 74 (100), 55 (35), 45 (10). The 1 H NMR spectrum of component A showed a basic similar pattern to those of well-known glycolipids but had significant variations. In the fraction A, there were only three prominent peaks at 1.143, 1.212 and 1.103 ppm. The former peak was assigned as the H-1 proton, and the latter peak was estimated to be the proton bound to the esterified carbon in the sugar backbone (data not shown). In addition, there was one signal at peak at 2.0 ppm (derived from an acetyl group). Furthermore, the number of protons at 0.9 ppm (–CH3 ) was about three. These results strongly indicated that only one fatty acyl group exists in the structure of component B. The results obtained from the above analyses clearly indicated that the surface active compound produced by MSA04 was a glycolipid derivative (Table 3). The average surface tension of pure water at 24 ◦ C was 72.54 ± 0.74 mN/m, SDS (0.3 mg/ml) showed the lowering surface tension activity to 30.12 ± 0.26 mN/m and the purified compound (0.2 mg/ml) showed the lowering surface tension activity to 16.34 ± 0.65 mN/m. The surfactant concentration in the extract was 16 g/l. 3.7. Stability of purified biosurfactant The biosurfactant produced by the strain MSA04 was stable over a range of temperatures particularly stable even after autoclaving (121 ◦ C). The biosurfactants were invariably stable over pH 5–9. The stability was high at alkaline pH compared to lower pH values. Considering the marine strains, NaCl concentration was inevitable

The hydrocarbon degradation potential was analysed based on the cell surface hydrophobicity. Interestingly, the cell hydrophobicity in the surfactant system decreased with surface active compound concentration (Fig. 8). In the present study, the strain N. lucentensis MSA04 showed hydrocarbon degradation potential. The comparison of the hydrophobicity in the hydrocarbon–surfactant system indicated that the hydrophobicity of surface cells in the hydrocarbon–surfactant system was the highest for N. lucentensis MSA04 (85%). The hydrophobicity of the N. lucentensis MSA04 was invariably consistent on kerosene, diesel and hexane. 3.9. Detection of biosynthetic genes in N. lucentensis MSA04 It was established that the rhamnolipid biosurfactant synthesis in Pseudomonas sp. is mediated by rhl genes and lipopeptide biosurfactant synthesis in Bacillus sp. is mediated by sfp gene. But in the present study, no PCR amplified product was detected for rhl and sfp genes, which ultimately evidenced unique biosynthetic pathway in N. lucentensis MSA04. However, the pks type II domain ketosynthase was detected in MSA04. The sequence was deposited in GenBank, EMBL in Europe and the DNA Data Bank of Japan with an accession number GQ153946. The tBLASTx program showed the partial pks gene cds of MSA04 as a putative ketasynthase domain of Streptomyces taxa. In variably all the sequences showed neighboring clustering with Streptomyces sp. Particularly N. lucentensis MSA04 showed clustering with beta ketosynthase domain of S. cinnamonensis A3823.5 (data not shown).

Fig. 7. The mass spectrum of hydrophobic part (nonanoic acid methyl ester) MSA04.

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4. Discussion Sponges offer nourishment and a safe habitat to their symbionts; it holds more nutrient than seawater and sediments [35,36]. Marine microorganisms are good candidates for bioremediation, pharmaceutical, and bioactive natural products. Association of culturable actinobacteria including Micromonospora and Streptomyces in the marine sponge Haliclona sp. have been reported earlier [37]. Micromonospora sp. has been isolated previously from a marine sponge Hymeniacidon perleve by Zhang et al. [38]. Maldonado et al. [39] described that the “Micromonospora–Rhodococcus–Streptomyces” group seems to be ubiquitous in cultured actinobacteria from marine environments. However, in the present study, no cardioforms appeared to be most dominant group among the cultured actinobacteria retrieved from the marine sponge D. nigra. The strain N. lucentensis MSA04 chosen in this study was based on its potential surfactant production as per the index of different screening methods used in the present study. The nocardioform actinomycetes in general play a crucial role in the degradation of PAHs in soils [40]. Although interest in biosurfactants is increasing, these compounds are not competitive with synthetic surfactants owing to high production costs. The use of alternative substrates (substitutes for conventional media), usually renewable resources, could be explored since substrates account for 50% of final product costs. Therefore, in the present study, the due emphasis was given for the development of chief raw materials for process optimization. Agro-industrial waste such as wheat bran, ground nut oil cake, rice bran, and oil seed cake and industrial waste such as furnished leather powder, diesel contaminated soil, petrol bunk soil, aavin (milk processing waste) pretreated sludge, aavin (milk processing waste) treated waste, tannery pretreated sludge, treated molasses (distillery waste), pretreated molasses (distillery waste), and tannery treated sludge were screened for SSC development. Based on the screening, the industrial and agro-industrial wastes such as oil seed cake, wheat bran, tannery treated sludge, tannery pretreated sludge, treated molasses and pretreated molasses were selected for SSC optimization. The highly preferred substrate for the strain MSA04 was wheat bran. The potential of industrial and agro-industrial wastes in the development of SSC for the production of biosurfactant from marine actinobacterium was demonstrated for the first time. Industrial effluents have recently shown good promise as potential substrates for biosurfactant production. Striking recent developments in this area have been reviewed by Desai and Banat [5]. Koch et al. [41] constructed Pseudomonas aeruginosa lacking the capacity to utilize lactose present in whey waste, by transferring the lac plasmid from Escherichia coli to P. aeruginosa, capable of rhamnolipid biosurfactant production from whey. It reported that the emulsification activity of the biosurfactant was not substrate-specific. However, substrate-specific emulsification by biosurfactant has been demonstrated by Falatko and Novak [42], showing that biosurfactants produced from growth on glucose or vegetable oil could not emulsify gasoline hydrocarbons, while biosurfactants produced from growth on gasoline could emulsify. Albeit the findings of the present study evidenced a substratespecific production, the production was not arrested on any of the tested substrates; instead, the production was drastically declined. Some microbes have been reported to be capable of producing surfactants with the ability to emulsify various hydrocarbons, irrespective of the substrates used as carbon source [43]. Among the metal ions screened, CuSO4 was shown to be the most appropriate metal ion for the production of biosurfactant by marine actinobacterium MSA04 followed by FeCl3 . One of the significant findings of the present study includes the production of biosurfactant in SSC supplemented with 20% (w/w) metal ions. Such high concentration of metal ions has never been reported to

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have a positive effect on the biosurfactant production. The findings of present study envisaged that the marine actinobacterium can be used for the bioremediation of heavy metal contaminated marine environment. Among the various solvent systems including acetone, dichloromethane, ethyl acetate, water and chloroform:methanol (2:1) used for the extraction of surface active compounds, the chloroform:methanol (2:1) was found to be highly effective solvent system. The recovery and purification of biosurfactants from complex fermentation broth is a major problem in the commercialization of biosurfactants. In many cases, the downstream process increases the cost of biosurfactant production to as high as 60% [5,44]. Thus, improving product yield, low material costs and combining the steps of recovery can reduce the recovery costs. In present study, the moistening media and the extraction protocol was newly developed for the biosurfactant production and downstream processing under SSC. Interestingly, kerosene was found to be the most suitable carbon source for the biosurfactant production by MSA04. Among the carbon sources screened, kerosene was shown to be the cheapest carbon source and further, it increases the scope of biosurfactant production using crude oil as carbon source, which ultimately attain the goal of hydrocarbon degradation. The hydrophobicity of the bacterial cell surface was tested using the BATH assay. The hydrophobicity of the cells grown in hydrocarbon-containing media was higher than cells grown in water-soluble substrate. This indicates the biosurfactant not only helps in emulsification but also plays a role in the change in the cell surface hydrophobicity to improve affinity of microbial cells for the substrate to facilitate their bioavailability [45]. Zhang and Miller [46] also suggested that the bioavailability of octadecane in the presence of rhamnolipid is controlled by both aqueous dispersion of octadecane and cell hydrophobicity. To obtain biosurfactant-producing actinobacteria, oil-degraders were screened with three methods including surface and interfacial tension measurement, the oil displacement activity (ODA) test and the drop collapsing test [14,47]. Emulsification activity enhanced the biodegradation of hydrocarbons by increasing their bioavailability to the microbes involved. In the present study, amplification was observed from PKS II gene, but PKS I and NRPS did not produce PCR amplification. The absence of these amplicons might indicate that either the clone lacks NRPS or PKS I and/or the degenerate primers might not be suitable to amplify these genes. Another possibility is that most of the NRPS genes are involved in the biosynthesis of bioactive secondary metabolites, instead, they are involved in functions such as iron metabolism or quorum sensing [48]. The findings of the present study evidenced that the biosynthetic pathway of biosurfactant synthesis in the marine actinobacterium MSA04 was unique, which do not have any precedence with surfactive genes present in Pseudomonas sp. and Bacillus sp. It was established that the rhamnolipid biosurfactant synthesis in Pseudomonas sp. is mediated by rhl genes and lipopeptide biosurfactant synthesis in Bacillus sp. is mediated by sfp gene. Albeit, both genes were not amplified in the PCR using MSA04 DNA as template, the presence of PKS II gene in the strain MSA04 evidenced its biosynthetic potential of secondary metabolites. The results obtained from the chemical analyses evidenced that the surface active compounds in the present study would be a new glycolipid derivative. One of the significant findings of the present study was the thermostability of biosurfactant produced by MSA04. The biosurfactant formed was stable even at autoclaving (121 ◦ C). Such extreme stability was reported by Abdel-Mawgoud et al. [49] for the P. aeruginosa strain. The thermal stability of the biosurfactants increased the scope of its application in a broader perspective including at conditions where high temperatures prevail as in microbially enhanced oil recovery. Considering the need of halotolerant strains and biosurfactants for the bioremediation of oil

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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 actinobacteria was stable up to 5% NaCl. Chemical surfactants, however, are deactivated by 2–3% salt concentration [50]. The present study would be the first report showed the emulsification index of biosurfactant was higher than the synthetic surfactants such as SDS, tween20 and tween80. Therefore, it can be concluded that the biosurfactant produced by the marine actinobacterium is a potential source over the chemical surfactants, can be used in the bioremediation of contaminated marine environments. Acknowledgements GSK is thankful to CSIR, New Delhi for the award of Senior Research Fellowship. This paper is an outcome of CSIR project No. 38(1128)/06/EMR-II. References [1] E.V. Chandrasekaran, J.N. Bemiller, in: R.L. Whistler (Ed.), Methods in Carbohydrate Chemistry, Academic Press Inc., New York, 1980, p. 89. [2] L.D. Busnell, H.F. Haas, J. Bacteriol. 41 (1941) 653. [3] M. Givskov, J. Ostling, L. Eberl, P. Lindum, A.B. Christensen, G. Christiansen, S. Molin, Kjelleberg, J. Bacteriol. 180 (1998) 742. [4] N.G.K. Karanth, P.G. Deo, N.K. Meenanadig, Ferment. Sci. Technol. 77 (1999) 116. [5] J.D. Desai, I.M. Banat, Mol. Biol. Rev. 61 (1997) 47. [6] R.S. Makkar, S.S. Cameotra, Appl. Microbiol. Biotechnol. 58 (2002) 428. [7] M.E. Mercade, M.A. Manresa, J. Am. Oil Chem. Soc. 71 (1994) 61. [8] E. Haba, M.J. Espuny, M. Busquets, A. Manresa, J. Appl. Microbiol. 88 (2000) 379. [9] M. Nitschke, Biotechnol. Prog. 21 (2005) 1562. [10] M.C.O. Souza, I.C. Robetro, A.M.F. Milagres, Appl. Microbiol. Biotechnol. 52 (1999) 768. [11] Y.S. Park, S.W. Kang, J.S. Lee, S.I. Hong, Appl. Microbiol. Biotechnol. 58 (2002) 761. [12] F. Tarocco, R.E. Lecuona, A.S. Couto, J.A. Arcas, Appl. Microbiol. Biotechnol. 68 (2005) 481. [13] C. Xiong, C. Shouwen, Z. Ming, Y. Ziniu, Appl. Microbiol. Biotechnol. 69 (2005) 390. [14] J. Selvin, T. Thangavelu, G.S. Kiran, R. Gandhimathi, S.S. Priya, Helgoland Mar. Res. 63 (2009) 239. [15] N.H. Youssef, K.E. Dunacn, D.P. Nagle, K.N. Savage, R.M. Knapp, M.J. McInerney, J. Microbiol. Meth. 56 (2004) 339.

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