Dataset on controlled production of polyhydroxyalkanoate-based microbead using double emulsion solvent evaporation technique

A significant source of microplastics is from the usage of microbeads in the market since petrochemical plastic bead is a material used in cosmetic scrubs. A possible way to counteract the problem is by the substitution of synthetic plastic to natural biodegradable polymer. Polyhydroxyalkanoate (PHA) is a general class of thermoplastic microbial polymer and it is the best alternative to some petrochemical plastics due to its biodegradability. Some PHA has earned its way into cosmetic application due to its biocompatibility. This data article reports data on the development of biodegradable microbeads by using the double emulsion solvent evaporation technique. Our data describe the extraction of biopolymer from marine bacteria that was cultivated in shaken flask culture, removal of endotoxins using oxidizing agent, the production of microbeads using a peristaltic pump with a specific flowrate and silicon tubing, and the cytotoxicity of the microbeads.

a b s t r a c t A significant source of microplastics is from the usage of microbeads in the market since petrochemical plastic bead is a material used in cosmetic scrubs. A possible way to counteract the problem is by the substitution of synthetic plastic to natural biodegradable polymer. Polyhydroxyalkanoate (PHA) is a general class of thermoplastic microbial polymer and it is the best alternative to some petrochemical plastics due to its biodegradability. Some PHA has earned its way into cosmetic application due to its biocompatibility. This data article reports data on the development of biodegradable microbeads by using the double emulsion solvent evaporation technique. Our data describe the extraction of biopolymer from marine bacteria that was cultivated in shaken flask culture, removal of endotoxins using oxidizing agent, the production of microbeads using a peristaltic pump with a specific flowrate and silicon tubing, and the cytotoxicity of the microbeads. &

Value of the data
This data comprise the methodological data of developing biodegradable microbeads from a novel PHA-producer, Massilia haematophila UMTKB-2.
This data represent the use of automated technique instead of manual pipetting technique to produce controlled bead size with rapid, continuous and high reproducibility.
This data can be used in future product application for the betterment of our earth by reducing the accumulation of microplastic waste in the ocean.
This data can be used to replace synthetic plastic microbeads in the market that are manufactured and used in consumer products, such as cosmetic scrubs.
This data serve as a benchmark as the first report on the production of PHA-based microbeads that could be commercialized as a PHA-based dermal exfoliating scrub.

Data
This data article reports on the methods to derive optimized microbeads from poly(3-hydroxybutyrate) [P(3HB)] homopolymer produced by Bacillus megaterium UMTKB-1 and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) P(3HB-co-3HV) copolymer produced by Massilia haematophila UMTKB-2 using the double emulsion solvent evaporation technique. Fig. 1 shows the scanning electron microscope (SEM) images of the produced PHA-based microbeads. Table 1 represents the SEM images showing various sizes of the microbeads. The size of commercially available synthetic microbead ranges from 8 to 56 mm [1] while human skin pore size ranges from 250 to 500 mm [2]. The PHA-based microbead produced in this data article ranged from 10.1 to 140 mm with an average diameter of 38.44 mm, which is more compatible with human skin pore size than the microbead sizes reported by Mohamed and co-workers that ranged from 0.3 to 0.6 mm [3], and those of Murueva and co-workers that ranged from 0.7 to 2.6 mm [4]. The endotoxin level of polymer recovered by the chloroform extraction from the Gram-negative Massilia haematophila UMTKB-2 was recorded at 30.72 EU/g. A drastic decrease of endotoxin level to 0.24 EU/g was observed after pyrogen removal using oxidizing agent. In vitro cell culture was carried out using human keratinocyte cells (HaCaT) on  the P(3HB) microbeads to evaluate the cytotoxicity. There were no IC 50 value recorded on the HaCaT cells as shown in Fig. 2.

Experimental design, materials and methods
The strains used in this experiment were Massilia haematophila UMTKB-2, isolated from brackish water in Mengabang Telipot, Kuala Terengganu, Terengganu, Malaysia [5] and Bacillus megaterium UMTKB-1, isolated from the tissue sample of marine sponges collected from Pulau Langkawi, Kedah, Malaysia [6]. Bacillus megaterium UMTKB-1 was employed to synthesize homopolymer P(3HB), while Massilia haematophila UMTKB-2 was employed to synthesize copolymer P(3HB-co-3HV). The bacterial strains were streaked on nutrient rich (NR) agar routinely. Bacillus megaterium UMTKB-1 and Massilia haematophila UMTKB-2 were first cultured into a sterile NR medium using shaken flask cultivation method for 14 and 12 h respectively to activate the cells at 200 rotations per minute (rpm) until the mid-exponential growth phase, after which the growth phase was determined by measuring the  optical density of the bacterial culture at 600 nm. The biosynthesis, harvesting and recovery of P(3HB) was carried out according to Yatim and co-workers using sweet water [6], whereas the biosynthesis, harvesting and recovery of P(3HB-co-3HV) was performed according to Kiun and co-workers [5]. Inactivation and removal of endotoxins from the polymers were performed using hydrogen peroxide, after which the endotoxin levels were tested using E-TOXATE™ Kits (Sigma Aldrich) [7]. PHA microbeads were prepared by the double emulsion solvent evaporation technique with some modifications to obtain larger particles using automated technique [3,4]. Approximately 0.4 g PHA was dissolved in 10 mL of dichloromethane. The dissolved solution prepared for emulsification was continuously extruded at a flow rate of 1.62 mL/min, with a 3.2 mm silicone tubing using a Watson Marlow 101U/R peristaltic pump into 0.5% (w/v) of polyvinyl alcohol (PVA). It was then homogenized at 10,000 rpm using Ultra-Turrax T-25 digital homogenizer until the solvent had completely evaporated. All emulsions were continuously mixed mechanically for 24 h, until the remaining solvent had completely evaporated. The microbeads were extracted by suction filtration using 0.2 mm nylon-66 filter paper, washed three times with distilled water, and dried overnight for scanning electron microscope analysis. The size range of the microbeads was analyzed by using 20 homopolymer and 20 copolymer beads respectively. Human keratinocyte cell culture (HaCaT) was prepared by culturing in DMEM (Modified Eagle Medium), incubated with sodium pyruvate, penicillin-streptomycin and fetal bovine serum (FBS), then detached by trypsinization according to Chee and co-workers [8]. Cells were seeded at 1 Â 105 cells/mL in each well and were incubated at 37°C in 5% CO 2 for 24 h. The sample (P3HB-based microbeads) was diluted to 100 m g/mL. The diluted sample was transferred to 96-well flat bottom culture plate and left for incubation at 37°C in 5% CO 2 for 24 h. The cell viability was assayed with MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and the absorbance was read after 2 h at 570 nm.