Identification of Melanin producer
A total of 66 distinct bacteria were isolated from the marine sponge Tethya citrina on SYP-SW agar plates. All the isolates were screened for melanin production, with isolates WH001 55 and WH001 82 being identified as the best melanin producers. The strain WH001 55 was selected for further study based on its ability to produce higher levels of melanin of 6.2 mg/ml as opposed to 3.2 mg/ml which was produced by WH001 82. The isolate WH001 55 was identified as a rod shaped Gram-negative bacterium, which produced a light yellow colour when grown on SYP-SW agar. Based on the morphological and phylogenetic analysis (ME algorithm) and taxonomic affiliation (RDP-II), the isolate WH001 55 was identified as a Pseudomonas sp. (Figure 1). The 16 S rRNA sequence was deposited in Genbank with an accession number KX390669. The strain WH001 55 also produced a yellow colour with zone formation on tyrosine agar plates occurring after 48 h of growth, with colour formation progressively increasing and turning a dark brown after 144 h.
Production of melanin
The effect of various nutritional factors and incubation time, affecting cell biomass and melanin production in WH001 55 was assessed (Fig. 2). Maximum production of biomass (20 mg/ml) and melanin yield (6.2 mg/ml) was observed on the 6th day of incubation, with melanin production being concomitant with increased cell biomass (Fig. 2A). The requirement of tyrosine for enhanced melanin production was evaluated, and supplementation of the production medium with 1% tyrosine was found to yield maximum biomass (17 mg/ml) and melanin (6.5 mg/ml) (Fig. 2B). The effect of nutritional factors required for enhanced melanin production was subsequently assessed in production media supplemented with 1% tyrosine (Fig. 2C–F). Being a marine isolate, biomass levels in strain WH001 55 were consistently high in the media supplemented with 0.5–1.5% NaCl and decreased between 2% and 2.5%. Concomitant changes in melanin production were also observed, with maximum melanin production being observed in the production media supplemented with 0.5% NaCl (6.5 mg/ml), (Fig. 2C). Supplementation of the medium with various nitrogen sources tested did not have a marked effect on the cell biomass levels, however melanin yield was slightly increased with supplementation of nitrogen sources such as yeast extracts, with 1% yeast extract yielding 6.9 mg/ml melanin in the production media (Fig. 2D). Of the carbon sources tested, supplementation with 1% starch resulted in enhanced production of melanin (7.6 mg/ml) and cell biomass (18.9 mg/ml), (Fig. 2E). While higher levels of starch supplementation in the production media resulted in higher cell biomass levels, 1% starch was found to be the optimal level for melanin production in the medium (Fig. 2F). This optimized media was used for scale-up production of melanin in shake-flask cultures and subsequently used in the extraction and preparation of Nm and nanocomposite films.
Stability and characteristics of melanin
A strong peak characteristic of melanin was observed at 220 nm in the UV spectrum. Melanin was exposed to various temperatures ranging from between 40 and 100 °C and was found to be stable to both UV irradiation and high temperatures. The extracted melanin was readily soluble in hexane, water (pH above 7.0) and DMSO. Melanin was insoluble in organic solvents including ether, ethyl acetate, methanol and ethanol. TLC analysis showed a violet coloured spot corresponding to an Rf value of 0.68 related to the pigment melanin. Subsequent purification of this TLC spot following DEAE-Cellulose chromatography resulted in a melanin fraction which upon FT-IR analysis, showed a broad absorption at 3273 cm−1 (Fig. 3). This appears to indicate the presence of associated or polymeric - OH groups or N-H stretching vibrations of the carboxylic acid, and phenolic groups of melanin; while smaller bands at 2929 cm−1 and 2873 cm−1 and may be a result of stretching vibrations of the aliphatic C-H groups. The characteristic strong band at 1625 cm−1 can be attributed to vibrations of aromatic ring C = C of amide I, C = O and/or of COO- groups, while bands at ~1400 to 1500 cm−1 may be due to aliphatic C-H groups.
Characteristics of Nm
The particle size was determined for the control melanin produced by Pseudomonas sp. WH00155 and for the nanomelanin. The size distribution of particles of the control melanin were in the range of 122.4 nm (with a smaller intensity of 11.4% and width of 19.87) and 820.4 nm (with a larger intensity of 88.6% and width 261.5) with a mean diameter of 519.6. However, the Nm showed a single peak with particle size of 189.3 nm with 100% intensity with a mean of 208.7 nm (Fig. 4A). The diameter of the Nm particles was measured using an SEM micrograph, and these spherical shaped particles had a diameter of 100–140 nm (Fig. 4B,C).
The free radical scavenging effect of Nm was assessed using the DPPH assay, with different concentrations of Nm ranging from between 20 and 120 µg ml−1 being employed. The free radical scavenging activity increased at higher Nm concentrations. Nm exhibited a high free radical scavenging activity of 67.8% comparable with ascorbic acid (positive control) which showed a scavenging activity of 64.08% (Supplementary Figure S1A). The antioxidant activity increased with increased Nm concentrations and with an increase in reaction time (Supplementary Figure S1B). The antimicrobial properties of melanin and Nm was then assessed against a range of bacterial strains (Supplementary Figure S1C). Zones of inhibition were observed with (a) Staphylococcus aureus, (b) Escherichia coli, (c) Pseudomonas aeruginosa and (d) Bacillus subtilus sp. after 24 h of incubation, with Nm displaying slightly stronger antibacterial activity than the melanin. In the microtitre plate assay, Nm (30 µg/ml) showed effective inhibition of S. aureus followed by E. coli, P. aeruginosa and B. subtilis (Supplementary Figure S1D), whereas melanin (80 µg/ml) showed a medium level of antibacterial activity against S. aureus followed by E. coli, P. aeruginosa and B. subtilis. Based on the MBC:MIC ratio, the mode of action of Nm was classified as “bactericidal” (Supplementary Tables S1 and S2). The antimicrobial activity was effected on both Gram negative and Gram positive bacteria and therefore Nm was grouped under broad-spectrum antibacterial compound.
Characteristics of Nm-PHB film
The moisture content of the Nm-PHB films dried overnight at 105 °C was 15.24 ± 0.253 (Nm: PHB: glycerol 1:1:1% w/v), 16.01 ± 0.423 (Nm: glycerol: PHB 2:1:1% w/v) and 18.20 ± 0.145 (Nm: glycerol: PHB 1:2:1% w/v) respectively. Visual observation of the films revealed characteristic features as shown in Fig. 5A,B and C. The equal proportion blend (PHB:glycerol:Nm 1:1:1% w/v) displayed a smooth surface appearance and was flexible, whereas the increased Nm concentration of 2% in the blend imparted a brown colour on the film and was more brittle. The film prepared using a blend of Nm:PHB:glycerol (1:2:1% w/v) displayed a rough surface together with a hard texture. SEM image analysis showed a smooth surface with minute cracks in the nanocomposite film prepared with a composition of Nm:PHB:glycerol (2:1:1% w/v) (Fig. 5D), whereas the film prepared using increased PHB, Nm:PHB:glycerol (1:2:1% w/v) showed roughness, highly compact and agglomerated structures (Fig. 5E). This may be due to the high crystalline nature of PHB. The nanocomposite film prepared with equal proportions of PHB: glycerol: Nm (1:1:1% w/v) under different magnifications such as 10 µm and 5 µm revealed a smooth surface morphology. The roughness was reduced and flexibility was improved in the equal proportion nanocomposite film (Fig. 5F and G). Thus the Nm appeared to impart a smooth, homogenous and flexible nature to the nanocomposite film prepared using an equal proportion of the components. The analysis also revealed that glycerol was an effective plasticizer blended in the Nm-PHB film.
XRD and AFM analysis of nanocomposite film
The crystallinity of the nanocomposite film was determined by X-ray diffraction (XRD) analysis (Fig. 6A). Peaks corresponding to the nanocomposite film prepared using Nm:PHB:glycerol (1:2:1% w/v) are shown in green (F1), while film prepared using PHB:glycerol:Nm (1:1:1% w/v) is shown in black (F2). In general, both peaks correspond to orthorhombic crystal planes (020), (110), (101) (111) (130) and (040) at 2θ values. Two high intensity sharp peaks were observed at around 13.5°, 16.8° and half width large, while small intensity peaks were detected near to 23.5°, and 24.5°. Other peaks of much smaller intensity were observed at 2θ values of (130) and (040) were 25.6° and 27°. Near to 2θ of 20°, a ß form of crystal was observed. In addition to this, several 2θ values were obtained at 32.6°, 37.6° and 46.3°. The lattice parameter was found to be similar for the film prepared using increased PHB (2%) and equal proportion of the Nm composite film (1:1:1). However, the intensity of the peaks varied with the concentration of PHB in the film. The intensity of the peak was slightly smaller in 1% PHB when compared to film prepared using 2% PHB. In general, melanin is an amorphous polymer whereas PHB is a thermostable crystalline polymer. The broad peaks observed in the XRD analysis depicts an amorphous state while the sharp peaks depict a crystal phase. It can be noted that the film prepared using increased Nm (2%) with 1% each of PHB and glycerol which are represented in red peaks (F3) showed a wider peak with a decrease in intensity ranging from 20.2° to 26.8°, and another peak at 32.6° and 46.3°. The wider peak indicates that increases in Nm concentrations in the blend leads to decreases in the crystallinity. The NanoScope analysis of AFM images (Fig. 6B and C) confirmed nanoscale surface roughness of the film (12.4 nm). The route mean squared (RMS) value is a measure of surface roughness. The AFM imaged confirmed the film was smooth, and without cracks. The AFM data analysis revealed the smooth homogenous surface of the film imparted by Nm.
Differential Scanning Calorimetry (DSC)
The DSC of melanin (control) (Fig. 7A), showed an endothermic peak at 68.68 °C. This may be due to the moisture content present in the sample with an onset temperature of 73.0 °C and 11.21 J/g of latent energy. A second degradation peak appeared in the range of 174.02 °C with an onset temperature of 160.74 °C and 26.69 J/g of latent energy (Fig. 7A). Film blended with PHB, Nm and glycerol (1:1:1 ratio) showed an endothermic peak at a temperature of 110.66 °C which again may be due to the moisture present in the film with an onset temperature of 78.56 °C and a latent energy of 188.5 J/g. A second small peak occurred in the region of 176 °C with an onset temperature of 167.66 °C and latent energy of 5.809 J/g (Fig. 7B). The second peak may belong to the melanin when compared with the control peak in the range of 174.02 °C. A small degradation peak at 265 °C may belong to the glycerol present in the prepared film. The degradation peak that occurred in the range of 292.94 °C with latent energy of 56.01 J/g may belong to PHB, a highly thermostable crystalline polymer. The DSC based experiments revealed that Nm blended with PHB can produce a highly thermo stable crystalline polymer.
Antibiofilm activity of Nm-PHB composite/film
The effect of Nm:PHB composite on biofilm formation was determined quantitatively in a microtiter plate assay (Fig. 8). Nm at varying concentration between 10 and 30 µg/ml showed antibiofilm activity against S. aureus. The 30 µg/ml Nm treated wells showed a 71% inhibition of the S. aureus biofilm. A complete inhibition of S. aureus biofilm was observed in the wells treated with Nm:PHB composite. These wells showed no indication of biofilm formation and were similar to the negative control. The antibiofilm activity was confirmed using confocal laser scanning microscope (CLSM) images with a glass slide / film surface biofilm assay (Fig. 8). The MDR S. aureus strain was allowed to form a biofilm on the control surfaces include a glass slide and µ slide (ibidi polymer made of high quality plastic) and on the test surface (Nm-PHB film). The control surfaces showed 100% coverage of a thick dense, clump of MDR S. aureus biofilm (Fig. 9A and B). The Nm-PHB film effectively inhibited the formation of a MDR S. aureus biofilm, as shown in a CLSM image (Fig. 9C). The Nm-PHB film resulted in complete protection against bacterial colonization, as the CLSM image showed no indication of biofilm formation on the surface of the film.
AbstractThis study reports on the development of bio-based hydrophobic coatings for packaging papers through deposition of polyhydroxybutyrate (PHB) particles in combination with nanofibrillated cellulose (NFC) and plant wax. In the first approach, PHB particles in the micrometer range (PHB-MP) were prepared through a phase-separation technique providing internally-nanosized structures. The particles were transferred as a coating by dip-coating filter papers in the particle suspension, followed by sizing with a carnauba wax solution. This approach allowed partial to almost full surface coverage of PHB-MP over the paper surface, resulting in static water contact angles of 105°–122° and 129°–144° after additional wax coating. In the second approach, PHB particles with submicron sizes (PHB-SP) were synthesized by an oil-in-water emulsion (o/w) solvent evaporation method and mixed in aqueous suspensions with 0–7 wt % NFC. After dip-coating filter papers in PHB-SP/NFC suspensions and sizing with a carnauba wax solution, static water contact angles of 112°–152° were obtained. The intrinsic properties of the particles were analyzed by scanning electron microscopy, thermal analysis and infrared spectroscopy, indicating higher crystallinity for PHB-SP than PHB-MP. The chemical interactions between the more amorphous PHB-MP particles and paper fibers were identified as an esterification reaction, while the morphology of the NFC fibrillar network was playing a key role as the binding agent in the retention of more crystalline PHB-SP at the paper surface, hence contributing to higher hydrophobicity. View Full-Text
Keywords: polyhydroxybutyrate; nanofibrillated cellulose; paper coating; hydrophobicitypolyhydroxybutyrate; nanofibrillated cellulose; paper coating; hydrophobicity►▼ Figures
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).
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