Appl Biochem Biotechnol DOI 10.1007/s12010-014-1208-4
Production and Properties of a Surface-Active Lipopeptide Produced by a New Marine Brevibacterium luteolum Strain W. F. D. Vilela & S. G. Fonseca & F. Fantinatti-Garboggini & V. M. Oliveira & M. Nitschke
Received: 24 April 2014 / Accepted: 25 August 2014 # Springer Science+Business Media New York 2014
Abstract Microbial-derived surfactants are molecules of great interest due to their environmentally friendly nature and low toxicity; however, their production cost is not competitive when compared to synthetics. Marine microorganisms are exposed to extremes of pressure, temperature, and salinity; hence, they can produce stable compounds under such conditions that are useful for industrial applications. A screening program to select marine bacteria able to produce biosurfactant using low-cost substrates (mineral oil, sucrose, soybean oil, and glycerol) was conducted. The selected bacterial strain showed potential to synthesize biosurfactants using mineral oil as carbon source and was identified as Brevibacterium luteolum. The surfaceactive compound reduced the surface tension of water to 27 mN m−1 and the interfacial tension (water/hexadecane) to 0.84 mN m−1 and showed a critical micelle concentration of 40 mg L−1. The biosurfactant was stable over a range of temperature, pH, and salt concentration and the emulsification index (E24) with different hydrocarbons ranging from 60 to 79 %. Structural characterization revealed that the biosurfactant has a lipopeptide nature. Sand washing removed 83 % of crude oil demonstrating the potential of the biosurfactants (BS) for bioremediation purposes. The new marine B. luteolum strain showed potential to produce high surfaceactive and stable molecule using a low-cost substrate. Keywords Biosurfactants . Mineral oil . Marine bacteria . Brevibacterium
Introduction Biosurfactants (BS) are a structurally diverse group of surface-active molecules synthesized by microorganisms. Rhamnolipids from Pseudomonas aeruginosa, surfactin from Bacillus subtilis, emulsan from Acinetobacter calcoaceticus, and sophorolipids from Candida W. F. D. Vilela : M. Nitschke (*) Instituto de Química de São Carlos, Universidade de São Paulo, Caixa Postal 780, CEP 13560-970 São Carlos, SP, Brazil e-mail: [email protected] S. G. Fonseca : F. Fantinatti-Garboggini : V. M. Oliveira Divisão de Recursos Microbianos, Centro Pluridisciplinar de Pesquisas Químicas, Biológicas e Agrícolas, Universidade Estadual de Campinas, Caixa Postal 6171, CEP 13083-970 Campinas, SP, Brazil
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bombicola are examples of well-known microbial-derived surfactants [1]. Microbial surfactants are categorized by their chemical composition and microbial origin. Rosenberg and Ron [2] suggested that biosurfactants can be divided into low-molecular-mass molecules, which efficiently lower surface and interfacial tension, and high-molecular-mass polymers, which are more effective as emulsion stabilizing agents. The major classes of low-mass surfactants include glycolipids, lipopeptides, and phospholipids, whereas high-mass surfactants include polymeric and particulate surfactants. When compared to conventional synthetic surfactants, the biosurfactants have some peculiar characteristics such as higher biodegradability and lower toxicity, lower CMC and higher surface activity, superior ability to form molecular assembly and liquid crystals, biological activity, and stability to extreme conditions of pH, salinity, and temperature [1, 3, 4]. In addition, biosurfactants can be produced from renewable substrates by biotechnological processes. The properties exhibited by these biomolecules are the main reason for the increasing interest in their commercial exploitation. Biosurfactants can be utilized as emulsifiers, foaming, solubilizers, antimicrobial, and anti-adhesive in diverse industrial sectors ranging from environmental [5], food [6], and pharmaceutical [7]. Environmental concern that has been developed in the last years combined with consumers’ demand for natural products triggers attention to biosurfactants as alternative “green” molecules to synthetic surfactants. However, competitive production of biosurfactants is still restricted by the high cost of production [8], and in the last decades, concentrated efforts have been done to turn biosurfactant production economical viable comparatively to synthetic surfactants. One strategy to reduce costs and also to ensure the sustainability of the biosurfactant production process is the use of cheap alternative substrates. Two main classes of low-cost substrates have been proposed for microbial BS production: water-soluble substrates, such as molasses and starch-rich wastes, and water-immiscible substrates, such as oils, hydrocarbons, and edible oily wastes [8]. The marine environment is exceptionally diverse, and marine microorganisms are exposed to extremes in pressure, temperature, salinity, and nutrient availability. These distinct marine environmental niches are likely to possess highly diverse bacterial communities, possessing potentially unique biochemistry [9]. Thus, marine habitat provides an excellent opportunity to discover newer compounds of commercial importance such as biosurfactants and bioemulsifiers [10]. Although marine environment represents a rich reservoir of novel biomolecules, the production of biosurfactants by marine microbes is yet little explored. This work describes the screening and identification of a marine bacterium able to produce biosurfactant using mineral oil as carbon source. Production, properties, and potential applications of the surface-active molecule were also investigated.
Materials and Methods Microorganisms Fifty-eight bacterial isolates associated to marine invertebrates were collected at the north coast of São Paulo State, Brazil [11]. The maintenance of the isolates was performed by cryopreservation at −80 °C (10 % glycerol). Screening of Biosurfactant Producers Screening program was performed using a minimal medium containing 0.5 % NH4NO3 as nitrogen source. The carbon sources tested (sucrose, glycerol, mineral oil, and soybean oil)
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were added at 2.0 % concentration. The medium was prepared using artificial seawater containing (g L−1) the following: KBr 0.1, NaCl 23.48, MgCl2·6H2O 10.61, CaCl2·2H2O 1.47, KCl 0.66, SrCl2·6H2O 0.04, Na2SO4 3.92, NaHCO3 0.19, H3BO3 0.03, and distilled water 1,000 mL. The pH was adjusted to 7.0±0.2. Stock cultures were transferred to agar plates and incubated for 24 h at 30 °C. A loop of culture was inoculated in 125-mL Erlenmeyer flask containing 25 mL of medium and incubated on rotary shaker (150 rpm) at 30 °C. Samples were withdrawn at defined time intervals, centrifuged (8,000×g, 15 min), and the cellfree broth analyzed for surface activity. Drop-collapse technique [12] against lubricant oil (Luma L100) and surface tension by pendant drop method (KSV CAM 101) were evaluated for each bacterial culture growing on the different carbon sources proposed. Identification of Strain After bacterial growth on agar plate, genomic DNA of pure culture was isolated according to the protocol described by Pospiech and Neumann [13]. PCR amplification of 16S ribosomal DNA (rDNA) gene fragments was performed using the primers 27F [14] and 1401R [15], homologous to conserved regions of the 16S ribosomal RNA (rRNA) gene of the Bacteria domain. Fifty-microliter reaction mixtures containing 50–100 ng of genomic DNA, 2 U of Taq DNA polymerase (Invitrogen), 1× Taq buffer, 1.5 mM MgCl2, 0.2 mM of dNTP mix (GE Healthcare), and 0.4 μM each primer. The amplification program consisted of 1 cycle at 95 °C for 2 min, 30 cycles at 94 °C for 1 min, 55 °C for 1 min, 72 °C for 3 min, and 1 cycle of final extension at 72 °C for 3 min, in an Eppendorf thermal cycler. PCR amplification of 16S rRNA gene fragments was confirmed on 1 % agarose gel stained with Sybr Safe (Invitrogen). 16S rDNA fragments were further purified using mini-columns (GFX PCR DNA and Gel Band Purification Kit, GE Healthcare) and subjected to sequencing in an automated sequencer (MegaBase 500, GE Healthcare). The sequencing reactions were performed with the Kit DYEnamic ET Dye Terminator Cycle Sequencing Kit for MegaBace DNA Analysis Systems (GE Healthcare), according to the manufacturer’s specifications. Primers used for sequencing were 10F, 1100R [14], and 782R [16]. Partial 16S rRNA gene sequences obtained with each primer were assembled into a contig using phred/Phrap/CONSED program [17, 18]. Identification was achieved by comparing the contiguous 16S rRNA sequences obtained with sequence data from reference and type strains available in the public databases GenBank (www.ncbi.nlm.nih.gov) and Ribosomal Database Project (RDP Release 10; http://rdp. cme.msu.edu/). The sequences were aligned using the ClustalX program [19] and analyzed with MEGA software v.4 [20]. Evolutionary distances were derived from sequence-pair dissimilarities calculated as implemented in MEGA, using Kimura’s DNA substitution model [21]. The phylogenetic reconstruction was done using the neighbor-joining (NJ) algorithm [22], with bootstrap values calculated from 1,000 replicate runs. Kinetics of Biosurfactant Production Inoculum Preparation Stock culture of the bacterial strain was streaked to Marine Agar (Himedia) and cultivated for 24 h at 30 °C. A cell suspension was prepared to OD 0.5 ( 610 nm) and 1 mL transferred to 250-mL Erlenmeyer flasks containing 50 mL of Marine broth maintained at 30 °C, 150 rpm, 18 h. An aliquot of 4 mL of the actively growing cells were inoculated on 250-mL Erlenmeyer flask containing 46 mL of minimal medium (described above) with mineral oil (pharmaceutical grade) as the carbon source (2.0 %) and incubated at 30 °C on a rotary shaker
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(150 rpm) for 5 days. At regular intervals, samples were submitted to the following measurements: – –
Biomass estimation: cell growth was estimated by the protein content [23] of the culture Surface activity: culture samples were centrifuged at 8,000×g for 10 min and the surface tension (ST) of cell-free broth determined using a tensiometer (Attension Sigma 700) by the ring method. The critical micelle dilutions (CMD−1 and CMD−2) were measured as the surface tension of 10 and 100 times dilutions of the broth in distilled water.
Biosurfactant Isolation The biosurfactant was isolated from cell-free broth by acid precipitation after adjusting the pH to 2.0 using HCl and keeping it at 4 °C overnight. The precipitate thus obtained was pelleted at 10,000×g for 20 min and dried at 50 °C. The crude surfactant obtained was utilized for the subsequent tests. Surface Properties of the Biosurfactants Surface tension of aqueous solutions of the BS (0.1 %) was measured as described above. Interfacial tension was performed against hexadecane. The critical micelle concentration (CMC) was automatically determined by measuring the surface tension of serial dilutions of the BS and calculated using OneAttension Software. Stability Studies Thermal stabilities of BS solutions were determined after incubation at different temperatures (−4 °C to 100 °C) for 24 h and cooling to room temperature. The stability to sterilization (121 °C/20 min) was performed using an autoclave. The pH stability was performed by adjusting solution to different pH values using 1 N NaOH or HCl. For studying the effect of salt addition on surfactant activity, different concentrations of NaCl were added and mixed until complete dissolution. The surface tension measurement was performed as described above. Emulsifying Activity Aliquots of 6 mL of each hydrocarbon were added to 4 mL of surfactant solution (0.1 % w/v) and vortexed at high speed for 2 min. After 24 h, the emulsification index (E24) was calculated dividing the measured height of emulsion layer by the mixture’s total height and multiplying by 100 [24]. Crude Oil Removal The biosurfactant solution was used to wash sand samples contaminated with crude oil. Low viscosity crude oil was added (10 % w/w) to the sand (≈65 mesh) and left at room temperature for 7 days. Then, 5 g of sand samples were added of 20-mL biosurfactant solution (0.1 %) and put in a rotary shaker at 200 rpm for 6 h at 30 °C. The washing was repeated, aqueous solutions decanted, and the sand dried at 50 °C for 12 h. Water washing was used as control.
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The residual oil present in sand was extracted with dichloromethane (2×10 mL), and the solvent was evaporated at 50 °C. The remaining oil was determined gravimetrically and the % of oil removal was calculated using the following equation: Crude oil removedð%Þ ¼ ðOi−OrÞ=Oi 100% where Oi is the initial oil in the sand (g) before washing, and Or is the oil remaining in the sand (g) after washing [25]. Preliminary Structural Characterization Thin-Layer Chromatography Qualitative thin-layer chromatography (TLC) was performed using silica gel (Alugram® Xtra Sil G/UV254) plates and the solvent mixture acetonitrile/methanol/n-butanol/water (60:20:5:2). The resultant spots on the TLC plates were visualized by spraying of Molisch reagent for carbohydrates and ninhydrin for protein. The TLC plates were exposed in an iodine chamber to visualize the lipid fractions. Infrared Spectroscopy The infrared (IR) analysis of BS was recorded on a Fourier transform infrared (FTIR) spectrophotometer (Bomem/MB 102). The samples were dispersed in KBr pellets, and the spectra were obtained from 32 scans with a resolution of 4 cm−1 in the range of 500– 4,000 cm−1. Statistics The experiments are expressed as the mean of at least three independent replicates. Error bars (when shown) represent standard deviation.
Results and Discussion Bacterial Selection and Identification The screening program to identify marine bacteria able to produce surface-active compounds using low-cost carbon sources resulted in the preliminary selection of 12 isolates that reduced the surface tension of media to levels below 40 mN m−1 and/or showed positive results on drop collapse test. The isolates demonstrated preference to produce surface-active compounds when hydrophobic carbon sources (mineral oil and soybean oil) were present on medium; probably those substrates stimulates BS synthesis increasing bioavailability of the hydrophobic insoluble nutrients to the cells [26]. Among all marine bacteria tested, the isolate named AC189a showed the lowest surface tension when mineral oil was the substrate (Table 1) and was selected for further studies. Note that when growing on soybean oil, no reduction on surface tension was detected suggesting that the presence of an insoluble carbon source is not essential for BS synthesis. Possibly when growing in triglyceride-rich substrates, the bacteria can express lipases [27] that enhance solubility of the oil without the need of a surface-active agent. Ferhat et al. [28] have also evaluated biosurfactant production using glucose, olive oil,
Appl Biochem Biotechnol Table 1 Selection of low-cost carbon sources for BS production by the marine Brevibacterium luteolum strain Surface tension (mN m−1) and drop-collapse testa
Substrate
Time (h) 24
32
40
48
56
64
72
80
88
96
Soybean oil
68.2
71.0
70.1
59.8
70.4
70.4
70.7
51.3
53.1
71.1
Mineral oil Sucrose
68.2 69.3
70.6 71.3
71.3 71.0
71.2 70.6
70.8 70.8
70.8 69.7
35.5c 70.6
28.9c 70.9
29.1c 70.8
38.3d 70.9
Glycerol
68.7
70.8
70.7
70.9
70.9
70.2
70.9
70.7
70.6
71.0
a
Activity showed when present: a (+), b (++), c (+++), d (++++)
hexadecane, and crude oil as carbon sources and reported that the best results were obtained using hexadecane. Blast-n search revealed that the isolate AC189a has 99 % sequence similarity with 16S rRNA gene sequences from several B. luteolum strains available at the public database GenBank (http://www.ncbi.nlm.nih.gov/), including the type strain. Phylogenetic reconstruction based on the 16S rRNA gene allowed the recovery of the isolate AC189a in a tight cluster, supported by a high bootstrap value (90 %), with the type strain of B. luteolum, defining the identification of the isolate at the species level (Fig. 1). This bacterium was isolated from the ascidian Didemnum ligulum and the 16S rRNA sequence determined for AC189a was deposited at the GenBank database under the accession number JN615454. Biosurfactant Production Figure 2 illustrates the time course of biosurfactant production by B. luteolum using 2 % mineral oil as sole carbon source. The maximal reduction on surface activity was detected when the culture reached stationary phase (about 60 h) subsequently, an increase was observed on surface tension and CMD values that can be relative to some BS degradation due to low
T
100 Brevibacterium sanguinis DSM15677 (AJ564859) 100
T
Brevibacterium celere DSM15453 (AY228463) T
Brevibacterium casei DSM20657 (AJ251418)
70
T
Brevibacterium otitidis DSM10718 (AF133534) T
100 Brevibacterium luteolum DSM15022 (AJ488509) 90 T
Brevibacterium ravenspurgense DSM21258 (EU086793) T
100 Brevibacterium massiliense JCM18108 (EU868814) T
Brevibacterium pityocampae DSM21720 (EU484189) T
Microbacterium schleiferi DSM20489 (Y17237) 0.01
Fig. 1 Phylogenetic analysis based on partial 16S rRNA sequences (∼1,000 bp) obtained from the isolate AC189a and related species. Bootstrap values (1,000 replicate runs, shown as %) greater than 70 % are listed. GenBank accession numbers are listed after species names. Microbacterium schleiferi DSM20489T was used as the outgroup
Appl Biochem Biotechnol biomass
pH
surface tension
-1
-2
CMD
CMD
8 70
7
60
50 40
50
30 40
20 10
30 0
10 0
20
40
60
80
100
120
6
ST, CMD (mN/m)
Biomass protein (µg/mL)
60
5
pH
70
4 3 2 1
Time (h)
Fig. 2 Time course of biosurfactant production by Brevibacterium luteolum using mineral oil as carbon source
stability or enzymatic activity [29]. The surface-active compound such produced was recovered from culture medium and submitted to further studies. Mineral oil is obtained as a byproduct of petroleum distillation and is a low-cost substrate that can be applied to BS production by B. luteolum. The ability to grow using mineral oil as the sole carbon source also suggest that the strain can be explored to biodegradation or to produce BS using other hydrocarbon substrates similar in composition to the mineral oil. Physicochemical Properties of Biosurfactant According to Haba et al. [30], a good surface-active agent should reduce surface and interfacial tension to values under 40.0 and 1.0 mN m−1, respectively. The biosurfactant obtained from the marine B. luteolum strain reduced the surface tension of water from 72.0 to 27.0 mN m−1 at the critical micelle concentration of 40.0 mg L−1 and reduce the interfacial tension waterhexadecane to 0.84 mN m−1. The results indicated that BS produced by B. luteolum possess high surface tension-lowering capacity and is both efficient and effective surfactant. The properties demonstrated by the isolated product are comparable to well-known BS such as surfactin and rhamnolipids. There are few reports in literature about BS from genus Brevibacterium; a study developed by Ferhat et al. [28] described that a biosurfactant isolated from a soil Brevibacterium sp. 7G presented a surface tension of 31.5 mN m−1 and a CMC of 2,000 mg L−1, whereas the purified surfactant obtained from Brevibacterium aureum MSA13 reduced the surface tension of water to 28.56 mN m−1 [31]. Stability Studies The applicability of biosurfactants in several fields depends on their stability at different temperatures, pH, and salt concentrations. Figure 3a shows that biosurfactant maintains its surface properties unaffected when submitted to temperatures between 25 and 60 °C for 24 h. Low
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a)
60
Surface Tension (mN/m)
50
40
30
20
10
0 -10
0
10
20
30
40
50
60
70
80
90 100 110 120 130
Temperature (°C)
b)
60
Surface Tension (mN/m)
50
40
30
20
10
0
2
4
6
8
10
12
pH
c)
60
50
Surface Tension (mN/m)
Fig. 3 Stability of B. luteolum surfactant under different temperatures (a), pHs (b), and NaCl concentrations (c)
40
30
20
10
0
0
5
10
% NaCl
15
20
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temperatures as 4 and −4 °C were included to simulate refrigeration of BS solutions. The data obtained demonstrated a loss on surface activity around 24 % when BS was maintained at −4 °C for 24 h. At 100 °C, surfactant stability was affected, increasing ST from 27.0 to 38.0 mN m−1, representing 40 % of loss. Autoclave sterilization (20 min) increased the surface tension from 27.0 to 30.4 mN m−1. Although the temperature affected BS stability, it is interesting to note that after heating at 100 °C for 24 h, the surface tension maintained below 40 mN m−1 was a useful characteristic when applying BS to industrial processes where heating treatments are important. The surface activity of the crude biosurfactant remained relatively stable to pH changes between pH 6 and 8, showing higher stability at alkaline than acidic conditions. At pH below 4, the BS showed significant increase on surface tension mainly due to the precipitation of the compound from solution, a fact also observed by several authors [32, 33]. At pH 12, the surface tension increased from 27.0 to 36.7 mN m−1 representing 36 % of loss (Fig. 3b). The effect of sodium chloride addition on biosurfactant produced from B. luteolum was studied. Little changes were observed in increased concentration of NaCl up to 16 % (w/v). At higher concentration of NaCl (up to 20 %), the biosurfactant retains 90 % of the surface tension (Fig. 3c). A biosurfactant produced by the marine B. aureum MSA13 was stable at pH ranging from 5 to 9, over NaCl concentrations from 1 to 5 % and to autoclave sterilization [31]. Another study have demonstrated that the BS produced by the isolate Brevibacterium sp. 7G was stable over 0.5– 10 % NaCl, at pH 4 to pH 11 and to temperatures from 20 to 100 °C [28]. The surfactant obtained in our study showed stability at high salinity and may be suitable for bioremediation in marine environment and also in industrial formulations with high salt concentrations. Once the bacterium was isolated from marine environment, a high stability to salt concentration was expected due to natural adaptation of the microorganism and its metabolites to these conditions. Emulsification Activity and Crude Oil Recovery The ability to form and stabilize emulsions is an important parameter to evaluate the quality of a surface-active agent. The BS was able to form stable emulsions with all hydrocarbons tested, showing E24 values from 60 to 79 % (Table 2), and the BS tend to form w/o emulsions. Many microbial surfactants are described to emulsify different hydrocarbons at different rates. Rhamnolipids from P. aeruginosa and surfactin form Bacillus subtilis were reported to emulsify and stabilize emulsions with various types of hydrocarbons and oils [34, 35]. Comparatively, a BS from Brevibacterium sp. 7G showed E24 of 95 % with motor oil and 55 % to sunflower oil [28]. Biosurfactants can emulsify hydrocarbons enhancing their water solubility, decreasing surface and interfacial tension, and increasing the displacement of oil from soil particles [5]; thus, they can be applied to remove petroleum pollutants by washing method [25]. The ability of biosurfactant obtained from B. luteolum to enhance crude oil removal from contaminated sand was also examined. Biosurfactant recovered 83 % of oil from contaminated sand, whereas when washing using distilled water, 17 % of recovery was obtained. The results suggests that biosurfactant may be applied in enhanced oil recovery, bioremediation of oil, and clean-up of oil tank; in addition, the ability to form emulsions with vegetable oil and fat suggests potential application as cleaning and emulsifying agent in food processing. Structural Characterization Preliminary chemical characterization of the surface-active compound was determined by TLC. A purple spot showing Rf 0.90 was visualized after staining with ninhydrin. With iodine vapor, yellowish spots with similar Rf values was showing. No spots were visualized after
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Table 2 Emulsification index of B. luteolum BS against different hydrocarbons
Hydrocarbon
E24 %
Hexadecane
64.0
Mineral oil Decane
60.0 70.5
Kerosene
79.3
Animal fat
74.0
Soybean oil
64.4
staining with Molisch reagent, indicating a lack of sugars in the sample. These results thereby indicated that the surface-active compound has a lipopeptide nature that was further confirmed by the IR. The IR spectra of BS (Fig. 4) showed an absorbance band with wave numbers ranging approximately from 3,400 cm−1. Absorbance in this region is caused as a result of C–H and N–H stretching vibrations and is a characteristic of carbon-containing compounds with amino groups. An absorbance in this region may also signify the presence of intramolecular hydrogen bonding. Three other absorbance peaks are seen at 2,854, 2,925, and 2,950 cm−1 signifying the presence of C–CH3 bonding or long alkyl chains. The peak in the spectrum at 1,735 cm−1 is %T
Fig. 4 FTIR spectroscopic analysis of the biosurfactant obtained from the marine Brevibacterium luteolum
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relative to carbonyl (C=O) stretching band. Absorbance in 1,656 cm−1 region signifies the presence of CO–N stretching vibrations. Other significant peak observed at 1,382 cm−1 corresponds to aliphatic chains (CH2). Peaks at 1,166 and 1,068 cm−1 are probably because of C–O vibrations in esters or in C–N aliphatic amines. The functional groups of biosurfactants were confirmed by FTIR spectra, which revealed the possible presence of amino, carbonyl, and alkyl groups. The preliminary characterization revealed that the marine bacteria B. luteolum is able to produce a lipopeptide biosurfactant when growing in mineral oil as sole carbon source. Biosurfactants derived from genus Brevibacterium have been recently described in literature; however, miscellaneous chemical compositions have been reported. Glycolipid biosurfactants were described to be produced by a marine Brevibacterium casei MSA19 [36] and by Brevibacterium sp. 7G isolated from hydrocarbon-contaminated soil [28]. Conversely, a marine B. aureum MSA13 strain was described to produce a lipopeptide surfactant [31]. According to the authors, the molecule isolated showed a hydrophobic moiety of octadecanoic acid methyl ester linked to the tetrapeptide sequence Pro-Leu-Gly-Gly and the newly BS was named brevifactin [31]. Lipopeptide surfactant family presents a wide range of useful properties to be explored in many fields. Surfactin obtained by Bacillus subtilis received this name due to its high surface activity [37] and is a typical lipopeptide biosurfactant. Apart from surface activity, surfactins and its derivatives as iturins and fengycins [38] display biological effects as anti-microbial, anti-tumor, anti-inflammatory, and anti-viral agents [39] that can be explored to development of new products. Our results demonstrated that B. luteolum also produces a lipopeptide surfactant, and further studies to elucidate the complete chemical structure of the molecule and its biological activity are under progress. The ability showed by the strain can be explored both to reduce BS production costs by using a cheap carbon source and also to degrade hydrocarbons similar in composition to mineral oil. To our knowledge, this is the first report on biosurfactant production by a B. luteolum strain.
Conclusion The screening of marine biosurfactant-producing microorganisms offers an excellent opportunity to the discovery of new molecules with distinctive properties. In this work, the marine strain B. luteolum was selected for its ability to grow and produce a potent surface-active molecule when growing in minimal marine medium using mineral oil as sole carbon source. The BS was stable over wide range of temperatures, pHs, and salt concentrations, formed stable emulsions with hydrocarbons and oils, and is suitable for bioremediation of crude oil. The preliminary chemical characterization revealed that the BS has a lipopeptide nature, and the surface properties presented by the BS indicate promising potential application in different fields, especially for environmental purposes. Acknowledgments Authors would like to thank FAPESP for financial support and to CAPES for fellowship. Authors also thank to Dr. Roberto Berlinck for providing the marine bacteria.
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Appl Biochem Biotechnol 3. Cameotra, S. S., Makkar, R. S., Kaur, J., & Mehta, S. K. (2010). Advances in Experimental Medicine and Biology, 672, 261–280. 4. Nitschke, M., & Costa, S. G. V. A. O. (2007). Trends in Food Science and Technology, 18, 252–259. 5. Pacwa-Płociniczak, M., Płaza, G. A., Piotrowska-Seget, Z., & Cameotra, S. S. (2011). International Journal of Molecular Sciences, 12, 633–654. 6. Nitschke, M., & Costa, S.G.V.A.O. (2014). In: C.N. Mulligan, S.K. Sharma, and A. Mudhoo (Eds), Biosurfactants: recent trends and applications (pp. 177–196). Boca Raton: CRC Press. 7. Gudina, E. J., Rangarajan, V., Sen, R., & Rodrigues, L. R. (2013). Trends Pharmacological Sciences, 34, 667–675. 8. Makkar, R. S., Cameotra, S. S., & Banat, I. M. (2011). AMB Express, 1, 5. 9. Kennedy, J., Marchesi, J. R., & Dobson, A. D. W. (2008). Microbial Cell Factories, 7, 27. 10. Satpute, S. K., Banat, I. M., Dhakephalkar, P. K., Banpurkar, A. G., & Chopade, B. A. (2010). Biotechnology Advances, 28, 436–450. 11. Menezes, C. B. A., Bonugli-Santos, R. C., Miqueletto, P. B., Passarini, M. R. Z., Silva, C. H. D., Justo, M. R., Leal, R. R., Fantinatti-Garboggini, F., Oliveira, V. M., Berlinck, R. G. S., & Sette, L. D. (2010). Microbiological Research, 165, 466–482. 12. Tugrul, T., & Cansunar, E. (2005). World Journal of Microbiology and Biotechnology, 21, 851–853. 13. Pospiech, A., & Neumann, B. (1995). Technical Tips, 11, 217–218. 14. Lane, D.J. (1991). In: M. Goodfellow and E. Stackebrandt (Eds), Nucleic acid techniques in bacterial systematics (pp. 115–147), New York: Wiley. 15. Heuer, H., Krsek, M., Baker, P., Smalla, K., & Wellington, E. M. (1997). Applied and Environmental Microbiology, 63, 3233–3241. 16. Chun, J. (1995), PhD thesis, University of Newcastle upon Tyne, England. 17. Ewing, B., Hillier, L., Wendl, M. C., & Green, P. (1998). Genome Research, 8, 175–185. 18. Gordon, D., Abajian, C., & Green, P. (1998). Genome Research, 8, 195–202. 19. Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F., & Higgins, D. G. (1997). Nucleic Acids Research, 24, 4876–4882. 20. Tamura, K., Dudley, J., Nei, M., & Kumar, S. (2007). Molecular Biology and Evolution, 24, 1596–1599. 21. Kimura, M. (1980). Journal of Molecular Evolution, 16, 111–120. 22. Saitou, N., & Nei, M. (1987). Molecular Biology and Evolution, 4, 406–425. 23. Lowry, O. H., Rosebough, N. J., Farr, A. L., & Randall, R. J. (1951). Journal of Biological Chemistry, 193, 265–275. 24. Iqbal, S., Khali, Z. M., & Malik, K. A. (1995). Letters in Applied Microbiology, 21, 176–179. 25. Urum, K., Pekdemir, T., & Copur, M. (2004). Journal of Colloids Interface Science, 276, 456–464. 26. Ron, E. Z., & Rosenberg, E. (2001). Environmental Microbiology, 3, 229–236. 27. Costa, S. G. V. A. O., Lépine, F., Milot, S., Déziel, E., Nitschke, M., & Contiero, J. (2009). Journal of Industrial Microbiology and Biotechnology, 36, 1063–1072. 28. Ferhat, S., Mnif, S., Badis, A., Eddouaouda, K., Alouaoui, R., Boucherit, A., Mhiri, N., Moulai-Mostefa, N., & Sayadi, S. (2011). International Biodeterioration and Biodegradation, 65, 1182–1188. 29. Nitschke, M., & Pastore, G. M. (2004). Applied Biochemistry and Biotechnology, 112, 163–172. 30. Haba, E., Espuny, M. J., Busquets, M., & Manresa, A. (2000). Journal of Applied Microbiology, 88, 379– 387. 31. Kiran, G. S., Thomas, T. A., Selvin, J., Sabarathnam, B., & Lipton, A. P. (2010). Bioresource Technology, 101, 2389–2396. 32. Chen, J., Huang, P. T., Zhang, K. Y., & Ding, F. R. (2012). Journal of Applied Microbiology, 112, 660–671. 33. Khopade, A., Biao, R., Liu, X., Mahadik, K., Zhang, L., & Kokare, C. (2012). Desalination, 285, 198–204. 34. Costa, S. G. V. A. O., Nitschke, M., Lépine, F., Déziel, E., & Contiero, J. (2010). Process Biochemistry, 45, 1511–1516. 35. Nitschke, M., & Pastore, G. M. (2006). Bioresource Technology, 97, 336–341. 36. Kiran, S., Sabua, A., & Selvin, J. (2010). Journal of Biotechnology, 148, 221–225. 37. Arima, K., Kakinuma, A., & Tamura, G. (1968). Biochemical and Biophysical Research Communications, 31, 488–494. 38. Ongena, M., & Jacques, P. (2008). Trends in Microbiology, 16, 115–125. 39. Seydlová, G., & Svobodová, J. (2008). Center Europe Journal Medicine, 3, 123–133.
Production and Properties of a Surface-Active Lipopeptide Produced by a New Marine Brevibacterium luteolum Strain W. F. D. Vilela & S. G. Fonseca & F. Fantinatti-Garboggini & V. M. Oliveira & M. Nitschke
Received: 24 April 2014 / Accepted: 25 August 2014 # Springer Science+Business Media New York 2014
Abstract Microbial-derived surfactants are molecules of great interest due to their environmentally friendly nature and low toxicity; however, their production cost is not competitive when compared to synthetics. Marine microorganisms are exposed to extremes of pressure, temperature, and salinity; hence, they can produce stable compounds under such conditions that are useful for industrial applications. A screening program to select marine bacteria able to produce biosurfactant using low-cost substrates (mineral oil, sucrose, soybean oil, and glycerol) was conducted. The selected bacterial strain showed potential to synthesize biosurfactants using mineral oil as carbon source and was identified as Brevibacterium luteolum. The surfaceactive compound reduced the surface tension of water to 27 mN m−1 and the interfacial tension (water/hexadecane) to 0.84 mN m−1 and showed a critical micelle concentration of 40 mg L−1. The biosurfactant was stable over a range of temperature, pH, and salt concentration and the emulsification index (E24) with different hydrocarbons ranging from 60 to 79 %. Structural characterization revealed that the biosurfactant has a lipopeptide nature. Sand washing removed 83 % of crude oil demonstrating the potential of the biosurfactants (BS) for bioremediation purposes. The new marine B. luteolum strain showed potential to produce high surfaceactive and stable molecule using a low-cost substrate. Keywords Biosurfactants . Mineral oil . Marine bacteria . Brevibacterium
Introduction Biosurfactants (BS) are a structurally diverse group of surface-active molecules synthesized by microorganisms. Rhamnolipids from Pseudomonas aeruginosa, surfactin from Bacillus subtilis, emulsan from Acinetobacter calcoaceticus, and sophorolipids from Candida W. F. D. Vilela : M. Nitschke (*) Instituto de Química de São Carlos, Universidade de São Paulo, Caixa Postal 780, CEP 13560-970 São Carlos, SP, Brazil e-mail: [email protected] S. G. Fonseca : F. Fantinatti-Garboggini : V. M. Oliveira Divisão de Recursos Microbianos, Centro Pluridisciplinar de Pesquisas Químicas, Biológicas e Agrícolas, Universidade Estadual de Campinas, Caixa Postal 6171, CEP 13083-970 Campinas, SP, Brazil
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bombicola are examples of well-known microbial-derived surfactants [1]. Microbial surfactants are categorized by their chemical composition and microbial origin. Rosenberg and Ron [2] suggested that biosurfactants can be divided into low-molecular-mass molecules, which efficiently lower surface and interfacial tension, and high-molecular-mass polymers, which are more effective as emulsion stabilizing agents. The major classes of low-mass surfactants include glycolipids, lipopeptides, and phospholipids, whereas high-mass surfactants include polymeric and particulate surfactants. When compared to conventional synthetic surfactants, the biosurfactants have some peculiar characteristics such as higher biodegradability and lower toxicity, lower CMC and higher surface activity, superior ability to form molecular assembly and liquid crystals, biological activity, and stability to extreme conditions of pH, salinity, and temperature [1, 3, 4]. In addition, biosurfactants can be produced from renewable substrates by biotechnological processes. The properties exhibited by these biomolecules are the main reason for the increasing interest in their commercial exploitation. Biosurfactants can be utilized as emulsifiers, foaming, solubilizers, antimicrobial, and anti-adhesive in diverse industrial sectors ranging from environmental [5], food [6], and pharmaceutical [7]. Environmental concern that has been developed in the last years combined with consumers’ demand for natural products triggers attention to biosurfactants as alternative “green” molecules to synthetic surfactants. However, competitive production of biosurfactants is still restricted by the high cost of production [8], and in the last decades, concentrated efforts have been done to turn biosurfactant production economical viable comparatively to synthetic surfactants. One strategy to reduce costs and also to ensure the sustainability of the biosurfactant production process is the use of cheap alternative substrates. Two main classes of low-cost substrates have been proposed for microbial BS production: water-soluble substrates, such as molasses and starch-rich wastes, and water-immiscible substrates, such as oils, hydrocarbons, and edible oily wastes [8]. The marine environment is exceptionally diverse, and marine microorganisms are exposed to extremes in pressure, temperature, salinity, and nutrient availability. These distinct marine environmental niches are likely to possess highly diverse bacterial communities, possessing potentially unique biochemistry [9]. Thus, marine habitat provides an excellent opportunity to discover newer compounds of commercial importance such as biosurfactants and bioemulsifiers [10]. Although marine environment represents a rich reservoir of novel biomolecules, the production of biosurfactants by marine microbes is yet little explored. This work describes the screening and identification of a marine bacterium able to produce biosurfactant using mineral oil as carbon source. Production, properties, and potential applications of the surface-active molecule were also investigated.
Materials and Methods Microorganisms Fifty-eight bacterial isolates associated to marine invertebrates were collected at the north coast of São Paulo State, Brazil [11]. The maintenance of the isolates was performed by cryopreservation at −80 °C (10 % glycerol). Screening of Biosurfactant Producers Screening program was performed using a minimal medium containing 0.5 % NH4NO3 as nitrogen source. The carbon sources tested (sucrose, glycerol, mineral oil, and soybean oil)
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were added at 2.0 % concentration. The medium was prepared using artificial seawater containing (g L−1) the following: KBr 0.1, NaCl 23.48, MgCl2·6H2O 10.61, CaCl2·2H2O 1.47, KCl 0.66, SrCl2·6H2O 0.04, Na2SO4 3.92, NaHCO3 0.19, H3BO3 0.03, and distilled water 1,000 mL. The pH was adjusted to 7.0±0.2. Stock cultures were transferred to agar plates and incubated for 24 h at 30 °C. A loop of culture was inoculated in 125-mL Erlenmeyer flask containing 25 mL of medium and incubated on rotary shaker (150 rpm) at 30 °C. Samples were withdrawn at defined time intervals, centrifuged (8,000×g, 15 min), and the cellfree broth analyzed for surface activity. Drop-collapse technique [12] against lubricant oil (Luma L100) and surface tension by pendant drop method (KSV CAM 101) were evaluated for each bacterial culture growing on the different carbon sources proposed. Identification of Strain After bacterial growth on agar plate, genomic DNA of pure culture was isolated according to the protocol described by Pospiech and Neumann [13]. PCR amplification of 16S ribosomal DNA (rDNA) gene fragments was performed using the primers 27F [14] and 1401R [15], homologous to conserved regions of the 16S ribosomal RNA (rRNA) gene of the Bacteria domain. Fifty-microliter reaction mixtures containing 50–100 ng of genomic DNA, 2 U of Taq DNA polymerase (Invitrogen), 1× Taq buffer, 1.5 mM MgCl2, 0.2 mM of dNTP mix (GE Healthcare), and 0.4 μM each primer. The amplification program consisted of 1 cycle at 95 °C for 2 min, 30 cycles at 94 °C for 1 min, 55 °C for 1 min, 72 °C for 3 min, and 1 cycle of final extension at 72 °C for 3 min, in an Eppendorf thermal cycler. PCR amplification of 16S rRNA gene fragments was confirmed on 1 % agarose gel stained with Sybr Safe (Invitrogen). 16S rDNA fragments were further purified using mini-columns (GFX PCR DNA and Gel Band Purification Kit, GE Healthcare) and subjected to sequencing in an automated sequencer (MegaBase 500, GE Healthcare). The sequencing reactions were performed with the Kit DYEnamic ET Dye Terminator Cycle Sequencing Kit for MegaBace DNA Analysis Systems (GE Healthcare), according to the manufacturer’s specifications. Primers used for sequencing were 10F, 1100R [14], and 782R [16]. Partial 16S rRNA gene sequences obtained with each primer were assembled into a contig using phred/Phrap/CONSED program [17, 18]. Identification was achieved by comparing the contiguous 16S rRNA sequences obtained with sequence data from reference and type strains available in the public databases GenBank (www.ncbi.nlm.nih.gov) and Ribosomal Database Project (RDP Release 10; http://rdp. cme.msu.edu/). The sequences were aligned using the ClustalX program [19] and analyzed with MEGA software v.4 [20]. Evolutionary distances were derived from sequence-pair dissimilarities calculated as implemented in MEGA, using Kimura’s DNA substitution model [21]. The phylogenetic reconstruction was done using the neighbor-joining (NJ) algorithm [22], with bootstrap values calculated from 1,000 replicate runs. Kinetics of Biosurfactant Production Inoculum Preparation Stock culture of the bacterial strain was streaked to Marine Agar (Himedia) and cultivated for 24 h at 30 °C. A cell suspension was prepared to OD 0.5 ( 610 nm) and 1 mL transferred to 250-mL Erlenmeyer flasks containing 50 mL of Marine broth maintained at 30 °C, 150 rpm, 18 h. An aliquot of 4 mL of the actively growing cells were inoculated on 250-mL Erlenmeyer flask containing 46 mL of minimal medium (described above) with mineral oil (pharmaceutical grade) as the carbon source (2.0 %) and incubated at 30 °C on a rotary shaker
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(150 rpm) for 5 days. At regular intervals, samples were submitted to the following measurements: – –
Biomass estimation: cell growth was estimated by the protein content [23] of the culture Surface activity: culture samples were centrifuged at 8,000×g for 10 min and the surface tension (ST) of cell-free broth determined using a tensiometer (Attension Sigma 700) by the ring method. The critical micelle dilutions (CMD−1 and CMD−2) were measured as the surface tension of 10 and 100 times dilutions of the broth in distilled water.
Biosurfactant Isolation The biosurfactant was isolated from cell-free broth by acid precipitation after adjusting the pH to 2.0 using HCl and keeping it at 4 °C overnight. The precipitate thus obtained was pelleted at 10,000×g for 20 min and dried at 50 °C. The crude surfactant obtained was utilized for the subsequent tests. Surface Properties of the Biosurfactants Surface tension of aqueous solutions of the BS (0.1 %) was measured as described above. Interfacial tension was performed against hexadecane. The critical micelle concentration (CMC) was automatically determined by measuring the surface tension of serial dilutions of the BS and calculated using OneAttension Software. Stability Studies Thermal stabilities of BS solutions were determined after incubation at different temperatures (−4 °C to 100 °C) for 24 h and cooling to room temperature. The stability to sterilization (121 °C/20 min) was performed using an autoclave. The pH stability was performed by adjusting solution to different pH values using 1 N NaOH or HCl. For studying the effect of salt addition on surfactant activity, different concentrations of NaCl were added and mixed until complete dissolution. The surface tension measurement was performed as described above. Emulsifying Activity Aliquots of 6 mL of each hydrocarbon were added to 4 mL of surfactant solution (0.1 % w/v) and vortexed at high speed for 2 min. After 24 h, the emulsification index (E24) was calculated dividing the measured height of emulsion layer by the mixture’s total height and multiplying by 100 [24]. Crude Oil Removal The biosurfactant solution was used to wash sand samples contaminated with crude oil. Low viscosity crude oil was added (10 % w/w) to the sand (≈65 mesh) and left at room temperature for 7 days. Then, 5 g of sand samples were added of 20-mL biosurfactant solution (0.1 %) and put in a rotary shaker at 200 rpm for 6 h at 30 °C. The washing was repeated, aqueous solutions decanted, and the sand dried at 50 °C for 12 h. Water washing was used as control.
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The residual oil present in sand was extracted with dichloromethane (2×10 mL), and the solvent was evaporated at 50 °C. The remaining oil was determined gravimetrically and the % of oil removal was calculated using the following equation: Crude oil removedð%Þ ¼ ðOi−OrÞ=Oi 100% where Oi is the initial oil in the sand (g) before washing, and Or is the oil remaining in the sand (g) after washing [25]. Preliminary Structural Characterization Thin-Layer Chromatography Qualitative thin-layer chromatography (TLC) was performed using silica gel (Alugram® Xtra Sil G/UV254) plates and the solvent mixture acetonitrile/methanol/n-butanol/water (60:20:5:2). The resultant spots on the TLC plates were visualized by spraying of Molisch reagent for carbohydrates and ninhydrin for protein. The TLC plates were exposed in an iodine chamber to visualize the lipid fractions. Infrared Spectroscopy The infrared (IR) analysis of BS was recorded on a Fourier transform infrared (FTIR) spectrophotometer (Bomem/MB 102). The samples were dispersed in KBr pellets, and the spectra were obtained from 32 scans with a resolution of 4 cm−1 in the range of 500– 4,000 cm−1. Statistics The experiments are expressed as the mean of at least three independent replicates. Error bars (when shown) represent standard deviation.
Results and Discussion Bacterial Selection and Identification The screening program to identify marine bacteria able to produce surface-active compounds using low-cost carbon sources resulted in the preliminary selection of 12 isolates that reduced the surface tension of media to levels below 40 mN m−1 and/or showed positive results on drop collapse test. The isolates demonstrated preference to produce surface-active compounds when hydrophobic carbon sources (mineral oil and soybean oil) were present on medium; probably those substrates stimulates BS synthesis increasing bioavailability of the hydrophobic insoluble nutrients to the cells [26]. Among all marine bacteria tested, the isolate named AC189a showed the lowest surface tension when mineral oil was the substrate (Table 1) and was selected for further studies. Note that when growing on soybean oil, no reduction on surface tension was detected suggesting that the presence of an insoluble carbon source is not essential for BS synthesis. Possibly when growing in triglyceride-rich substrates, the bacteria can express lipases [27] that enhance solubility of the oil without the need of a surface-active agent. Ferhat et al. [28] have also evaluated biosurfactant production using glucose, olive oil,
Appl Biochem Biotechnol Table 1 Selection of low-cost carbon sources for BS production by the marine Brevibacterium luteolum strain Surface tension (mN m−1) and drop-collapse testa
Substrate
Time (h) 24
32
40
48
56
64
72
80
88
96
Soybean oil
68.2
71.0
70.1
59.8
70.4
70.4
70.7
51.3
53.1
71.1
Mineral oil Sucrose
68.2 69.3
70.6 71.3
71.3 71.0
71.2 70.6
70.8 70.8
70.8 69.7
35.5c 70.6
28.9c 70.9
29.1c 70.8
38.3d 70.9
Glycerol
68.7
70.8
70.7
70.9
70.9
70.2
70.9
70.7
70.6
71.0
a
Activity showed when present: a (+), b (++), c (+++), d (++++)
hexadecane, and crude oil as carbon sources and reported that the best results were obtained using hexadecane. Blast-n search revealed that the isolate AC189a has 99 % sequence similarity with 16S rRNA gene sequences from several B. luteolum strains available at the public database GenBank (http://www.ncbi.nlm.nih.gov/), including the type strain. Phylogenetic reconstruction based on the 16S rRNA gene allowed the recovery of the isolate AC189a in a tight cluster, supported by a high bootstrap value (90 %), with the type strain of B. luteolum, defining the identification of the isolate at the species level (Fig. 1). This bacterium was isolated from the ascidian Didemnum ligulum and the 16S rRNA sequence determined for AC189a was deposited at the GenBank database under the accession number JN615454. Biosurfactant Production Figure 2 illustrates the time course of biosurfactant production by B. luteolum using 2 % mineral oil as sole carbon source. The maximal reduction on surface activity was detected when the culture reached stationary phase (about 60 h) subsequently, an increase was observed on surface tension and CMD values that can be relative to some BS degradation due to low
T
100 Brevibacterium sanguinis DSM15677 (AJ564859) 100
T
Brevibacterium celere DSM15453 (AY228463) T
Brevibacterium casei DSM20657 (AJ251418)
70
T
Brevibacterium otitidis DSM10718 (AF133534) T
100 Brevibacterium luteolum DSM15022 (AJ488509) 90 T
Brevibacterium ravenspurgense DSM21258 (EU086793) T
100 Brevibacterium massiliense JCM18108 (EU868814) T
Brevibacterium pityocampae DSM21720 (EU484189) T
Microbacterium schleiferi DSM20489 (Y17237) 0.01
Fig. 1 Phylogenetic analysis based on partial 16S rRNA sequences (∼1,000 bp) obtained from the isolate AC189a and related species. Bootstrap values (1,000 replicate runs, shown as %) greater than 70 % are listed. GenBank accession numbers are listed after species names. Microbacterium schleiferi DSM20489T was used as the outgroup
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pH
surface tension
-1
-2
CMD
CMD
8 70
7
60
50 40
50
30 40
20 10
30 0
10 0
20
40
60
80
100
120
6
ST, CMD (mN/m)
Biomass protein (µg/mL)
60
5
pH
70
4 3 2 1
Time (h)
Fig. 2 Time course of biosurfactant production by Brevibacterium luteolum using mineral oil as carbon source
stability or enzymatic activity [29]. The surface-active compound such produced was recovered from culture medium and submitted to further studies. Mineral oil is obtained as a byproduct of petroleum distillation and is a low-cost substrate that can be applied to BS production by B. luteolum. The ability to grow using mineral oil as the sole carbon source also suggest that the strain can be explored to biodegradation or to produce BS using other hydrocarbon substrates similar in composition to the mineral oil. Physicochemical Properties of Biosurfactant According to Haba et al. [30], a good surface-active agent should reduce surface and interfacial tension to values under 40.0 and 1.0 mN m−1, respectively. The biosurfactant obtained from the marine B. luteolum strain reduced the surface tension of water from 72.0 to 27.0 mN m−1 at the critical micelle concentration of 40.0 mg L−1 and reduce the interfacial tension waterhexadecane to 0.84 mN m−1. The results indicated that BS produced by B. luteolum possess high surface tension-lowering capacity and is both efficient and effective surfactant. The properties demonstrated by the isolated product are comparable to well-known BS such as surfactin and rhamnolipids. There are few reports in literature about BS from genus Brevibacterium; a study developed by Ferhat et al. [28] described that a biosurfactant isolated from a soil Brevibacterium sp. 7G presented a surface tension of 31.5 mN m−1 and a CMC of 2,000 mg L−1, whereas the purified surfactant obtained from Brevibacterium aureum MSA13 reduced the surface tension of water to 28.56 mN m−1 [31]. Stability Studies The applicability of biosurfactants in several fields depends on their stability at different temperatures, pH, and salt concentrations. Figure 3a shows that biosurfactant maintains its surface properties unaffected when submitted to temperatures between 25 and 60 °C for 24 h. Low
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a)
60
Surface Tension (mN/m)
50
40
30
20
10
0 -10
0
10
20
30
40
50
60
70
80
90 100 110 120 130
Temperature (°C)
b)
60
Surface Tension (mN/m)
50
40
30
20
10
0
2
4
6
8
10
12
pH
c)
60
50
Surface Tension (mN/m)
Fig. 3 Stability of B. luteolum surfactant under different temperatures (a), pHs (b), and NaCl concentrations (c)
40
30
20
10
0
0
5
10
% NaCl
15
20
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temperatures as 4 and −4 °C were included to simulate refrigeration of BS solutions. The data obtained demonstrated a loss on surface activity around 24 % when BS was maintained at −4 °C for 24 h. At 100 °C, surfactant stability was affected, increasing ST from 27.0 to 38.0 mN m−1, representing 40 % of loss. Autoclave sterilization (20 min) increased the surface tension from 27.0 to 30.4 mN m−1. Although the temperature affected BS stability, it is interesting to note that after heating at 100 °C for 24 h, the surface tension maintained below 40 mN m−1 was a useful characteristic when applying BS to industrial processes where heating treatments are important. The surface activity of the crude biosurfactant remained relatively stable to pH changes between pH 6 and 8, showing higher stability at alkaline than acidic conditions. At pH below 4, the BS showed significant increase on surface tension mainly due to the precipitation of the compound from solution, a fact also observed by several authors [32, 33]. At pH 12, the surface tension increased from 27.0 to 36.7 mN m−1 representing 36 % of loss (Fig. 3b). The effect of sodium chloride addition on biosurfactant produced from B. luteolum was studied. Little changes were observed in increased concentration of NaCl up to 16 % (w/v). At higher concentration of NaCl (up to 20 %), the biosurfactant retains 90 % of the surface tension (Fig. 3c). A biosurfactant produced by the marine B. aureum MSA13 was stable at pH ranging from 5 to 9, over NaCl concentrations from 1 to 5 % and to autoclave sterilization [31]. Another study have demonstrated that the BS produced by the isolate Brevibacterium sp. 7G was stable over 0.5– 10 % NaCl, at pH 4 to pH 11 and to temperatures from 20 to 100 °C [28]. The surfactant obtained in our study showed stability at high salinity and may be suitable for bioremediation in marine environment and also in industrial formulations with high salt concentrations. Once the bacterium was isolated from marine environment, a high stability to salt concentration was expected due to natural adaptation of the microorganism and its metabolites to these conditions. Emulsification Activity and Crude Oil Recovery The ability to form and stabilize emulsions is an important parameter to evaluate the quality of a surface-active agent. The BS was able to form stable emulsions with all hydrocarbons tested, showing E24 values from 60 to 79 % (Table 2), and the BS tend to form w/o emulsions. Many microbial surfactants are described to emulsify different hydrocarbons at different rates. Rhamnolipids from P. aeruginosa and surfactin form Bacillus subtilis were reported to emulsify and stabilize emulsions with various types of hydrocarbons and oils [34, 35]. Comparatively, a BS from Brevibacterium sp. 7G showed E24 of 95 % with motor oil and 55 % to sunflower oil [28]. Biosurfactants can emulsify hydrocarbons enhancing their water solubility, decreasing surface and interfacial tension, and increasing the displacement of oil from soil particles [5]; thus, they can be applied to remove petroleum pollutants by washing method [25]. The ability of biosurfactant obtained from B. luteolum to enhance crude oil removal from contaminated sand was also examined. Biosurfactant recovered 83 % of oil from contaminated sand, whereas when washing using distilled water, 17 % of recovery was obtained. The results suggests that biosurfactant may be applied in enhanced oil recovery, bioremediation of oil, and clean-up of oil tank; in addition, the ability to form emulsions with vegetable oil and fat suggests potential application as cleaning and emulsifying agent in food processing. Structural Characterization Preliminary chemical characterization of the surface-active compound was determined by TLC. A purple spot showing Rf 0.90 was visualized after staining with ninhydrin. With iodine vapor, yellowish spots with similar Rf values was showing. No spots were visualized after
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Table 2 Emulsification index of B. luteolum BS against different hydrocarbons
Hydrocarbon
E24 %
Hexadecane
64.0
Mineral oil Decane
60.0 70.5
Kerosene
79.3
Animal fat
74.0
Soybean oil
64.4
staining with Molisch reagent, indicating a lack of sugars in the sample. These results thereby indicated that the surface-active compound has a lipopeptide nature that was further confirmed by the IR. The IR spectra of BS (Fig. 4) showed an absorbance band with wave numbers ranging approximately from 3,400 cm−1. Absorbance in this region is caused as a result of C–H and N–H stretching vibrations and is a characteristic of carbon-containing compounds with amino groups. An absorbance in this region may also signify the presence of intramolecular hydrogen bonding. Three other absorbance peaks are seen at 2,854, 2,925, and 2,950 cm−1 signifying the presence of C–CH3 bonding or long alkyl chains. The peak in the spectrum at 1,735 cm−1 is %T
Fig. 4 FTIR spectroscopic analysis of the biosurfactant obtained from the marine Brevibacterium luteolum
Appl Biochem Biotechnol
relative to carbonyl (C=O) stretching band. Absorbance in 1,656 cm−1 region signifies the presence of CO–N stretching vibrations. Other significant peak observed at 1,382 cm−1 corresponds to aliphatic chains (CH2). Peaks at 1,166 and 1,068 cm−1 are probably because of C–O vibrations in esters or in C–N aliphatic amines. The functional groups of biosurfactants were confirmed by FTIR spectra, which revealed the possible presence of amino, carbonyl, and alkyl groups. The preliminary characterization revealed that the marine bacteria B. luteolum is able to produce a lipopeptide biosurfactant when growing in mineral oil as sole carbon source. Biosurfactants derived from genus Brevibacterium have been recently described in literature; however, miscellaneous chemical compositions have been reported. Glycolipid biosurfactants were described to be produced by a marine Brevibacterium casei MSA19 [36] and by Brevibacterium sp. 7G isolated from hydrocarbon-contaminated soil [28]. Conversely, a marine B. aureum MSA13 strain was described to produce a lipopeptide surfactant [31]. According to the authors, the molecule isolated showed a hydrophobic moiety of octadecanoic acid methyl ester linked to the tetrapeptide sequence Pro-Leu-Gly-Gly and the newly BS was named brevifactin [31]. Lipopeptide surfactant family presents a wide range of useful properties to be explored in many fields. Surfactin obtained by Bacillus subtilis received this name due to its high surface activity [37] and is a typical lipopeptide biosurfactant. Apart from surface activity, surfactins and its derivatives as iturins and fengycins [38] display biological effects as anti-microbial, anti-tumor, anti-inflammatory, and anti-viral agents [39] that can be explored to development of new products. Our results demonstrated that B. luteolum also produces a lipopeptide surfactant, and further studies to elucidate the complete chemical structure of the molecule and its biological activity are under progress. The ability showed by the strain can be explored both to reduce BS production costs by using a cheap carbon source and also to degrade hydrocarbons similar in composition to mineral oil. To our knowledge, this is the first report on biosurfactant production by a B. luteolum strain.
Conclusion The screening of marine biosurfactant-producing microorganisms offers an excellent opportunity to the discovery of new molecules with distinctive properties. In this work, the marine strain B. luteolum was selected for its ability to grow and produce a potent surface-active molecule when growing in minimal marine medium using mineral oil as sole carbon source. The BS was stable over wide range of temperatures, pHs, and salt concentrations, formed stable emulsions with hydrocarbons and oils, and is suitable for bioremediation of crude oil. The preliminary chemical characterization revealed that the BS has a lipopeptide nature, and the surface properties presented by the BS indicate promising potential application in different fields, especially for environmental purposes. Acknowledgments Authors would like to thank FAPESP for financial support and to CAPES for fellowship. Authors also thank to Dr. Roberto Berlinck for providing the marine bacteria.
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