Poly(3-hydroxybutyrate) Degradation by Bacillus infantis sp. Isolated from Soil and Identification of phaZ and bdhA Expressing PHB Depolymerase

Poly(3-hydroxybutyrate) (PHB) is a biodegradable and biocompatible bioplastic. Effective PHB degradation in nutrient-poor environments is required for industrial and practical applications of PHB. To screen for PHB-degrading strains, PHB double-layer plates were prepared and three new Bacillus infantis species with PHB-degrading ability were isolated from the soil. In addition, phaZ and bdhA of all isolated B. infantis were confirmed using a Bacillus sp. universal primer set and established polymerase chain reaction conditions. To evaluate the effective PHB degradation ability under nutrient-deficient conditions, PHB film degradation was performed in mineral medium, resulting in a PHB degradation rate of 98.71% for B. infantis PD3, which was confirmed in 5 d. Physical changes in the degraded PHB films were analyzed. The decrease in molecular weight due to biodegradation was confirmed using gel permeation chromatography and surface erosion of the PHB film was observed using scanning electron microscopy. To the best of our knowledge, this is the first study on B. infantis showing its excellent PHB degradation ability and is expected to contribute to PHB commercialization and industrial composting.


Introduction
Plastics are widely used in our daily lives and industries because of their excellent mechanical strength and thermal stability [1].As the amount of plastic used increases, environmental pollution due to landfilling and incineration of plastic waste has become a problem.As an alternative for waste treatment and pollution, research on biodegradable plastics that can be degraded by microorganisms is being actively conducted [2,3].Among these bioplastics, poly(3-hydroxybutyrate) (PHB), which is the most representative structure of polyalkanoates (PHAs), is biosynthetic and biodegradable only through microbial processes, and has high human body compatibility and potential applications in the medical field [4].However, owing to the low biodegradability of biodegradable plastics, it is impossible to recycle them, and they are discharged as general garbage rather than as separate discharges [5].Therefore, to commercialize biodegradable plastics, a method that can effectively degrade them within a short time is required [6,7].
Microorganisms known to degrade PHB include Bacillus spp., Microbulbifer spp., Ralstonia eutropha, and the genus Streptomyces [8][9][10].B. infantis among Bacillus spp.are found in various environments such as soil and sea, and have the ability to degrade azo dyes that are difficult to biodegrade [11].There are reports of strains known to have PHB-degrading abilities, but there are no reports on the specific activity and characteristics of these PHBdegrading strains [8].Biodegradation occurs easily when PHB is the only carbon source in an environment with limited nitrogen and carbon sources [12].Therefore, for effective PHB biodegradation, it is important to identify strains with excellent PHB degradation abilities under nitrogen-and carbon-limited conditions.
The PHB degradation pathway consists of the hydrolysis of PHB to the monomer 3-hydroxybutyrate by PHB depolymerase (PhaZ) and the conversion of 3-hydroxybutyrate to acetoacetate by 3-hydroxybutyrate dehydrogenase (BdhA), which is used for microbial metabolism [13,14].These two key enzymes are regulated by the phaZ and bdhA genes, respectively [15].Therefore, the PHB degradation ability can be inferred from the identification of microorganisms with phaZ and bdhA and further improvements in PHB degradation ability can be expected through gene cloning and overexpression [16].
In this study, we isolated microorganisms that effectively degraded PHB from soil.A halo assay was performed to examine the PHB degradation ability of the isolated microorganisms in a PHB double-layer plate [17].The phaZ and bdhA genes of the PHB-degrading enzymes in B. infantis were identified using polymerase chain reaction (PCR) in B. infantis.We aimed to measure the PHB film degradation rate in a mineral-rich medium with a limited nitrogen source under soil-like conditions and confirmed the excellent PHB-degrading ability of the isolated strain in mineral medium (MM) [17].The molecular weight changes and surface degradation of the PHB films were analyzed.Additionally, its degradability against other bioplastics was tested.Novel strains with excellent PHB degradation abilities and the identification of phaZ and bdhA using PCR suggest the possibility of commercializing PHB and its industrial benefits for effective PHB degradation.

Chemicals
All chemicals used in this study were of analytical grade.The chloroform, PHB powder, and PHB films used for the plates and degradation experiments were purchased from Sigma-Aldrich (USA).The PHB pellets used for gel permeation chromatography (GPC) analysis were purchased from Goodfellow (UK).

Isolation of PHB-Degrading Strains
Soil samples were collected from a depth of 10 cm below the surface of a garbage landfill in Sejong, Korea and rice fields in Suwon, Korea.Each soil sample (1 g) was diluted in 10 -1 , 10 -2 , or 10 -3 autoclaved distilled water (DW) and spread onto Luria-Bertani (LB) agar plates.The plates were incubated at 37°C for 1 d, and colonies with different morphological characteristics were isolated from each plate.The colonies were incubated in liquid medium for 1 d to prepare stocks containing 20% (w/v) glycerol and were stored at -70°C until further use.

Halo Assay: Bioplastic Double-Layer Plate
To screen for the PHB degradation strain, 10 g/l PHB powder was suspended in DW and autoclaved at 121°C for 15 min.After autoclaving, the PHB powder suspension was stirred overnight at 150-180 rpm.Autoclaved agar medium (20 ml) was then added to the plate.The PHB suspension was mixed with autoclaved medium containing 2% agar at a ratio of 1:1 and poured onto the top layer [17].Autoclaved paper discs were placed in the specific section of the PHB double layer plate and inoculated with 8 μl of culture medium.Cultivation was performed at 37°C for 1 d.To prepare suspensions of other bioplastics such as polybutylene adipate terephthalate (PBAT) and polybutylene succinate (PBS), 0.2 g of bioplastics were emulsified in 30 ml of dichloromethane (DCM).Next, 100 ml of DW was added and sonicated for 10 min using a VC 505 (Sonics & Materials, Inc., USA).After sonication, the solution was heated in a 60°C water bath to evaporate DCM and autoclaved at 121°C for 15 min [18].

16S rRNA Sequencing
Colonies forming halo zones on the PHB plates were identified at the species level using 16S rRNA sequencing, PCR amplification, and the primers 27F and 1492R.Partial sequences were obtained using Solgent (Korea) and compared to sequences in the National Center for Biotechnology Information (NCBI) GenBank database (https:// blast.ncbi.nlm.nih.gov/Blast.cgi)using BLASTN tools [19].

Identification of PHB Degradation Genes Using PCR
To identify phaZ and bdhA in strains with PHB degradation activity, universal primers for Bacillus species were designed for each gene.MEGA-X was used to align phaZ sequences from 29 Bacillus strains and bdhA sequences from 35 Bacillus strains.Among the aligned sequences, primers were designed by identifying the conserved regions common to most strains.Primer sequences used to identify phaZ using PCR were forward (5'-AATAAGTGTTGGAACTGGTTTGA-3') and reverse (5'-GTGTCGGTATTGTACATTCCTTTATC-3').The primer sequences used to identify bdhA by PCR were forward (5'-ATTGAAGAATTTCCTACAGAA-3') and reverse (5'-TGGAGTATCCACATAACCAGGGCA-3').The PCR conditions for phaZ and bdhA used in this study are listed in Table 1.The polymerase used for PCR was 2X Pfu PCR Smart Mix 1 (Solgent, Korea).PCR products were separated using electrophoresis at 100 mV for 30 min on 1.5% agarose gel containing 4 μl EcoDye DNA Staining Solution (Solgent) in TAE buffer (Bioneer, Korea).

Analysis of Physical Changes on the Surface of PHB Film
To observe surface changes on the PHB film after degradation, scanning electron microscopy (SEM) was used.The samples were collected after incubation, washed with DW, and dried in an oven.Then, the PHB sample was coated with gold at 5 mA for 120 s, and backscatter electron images were acquired using SEM (JSM-IT700HR, USA, Jeol) at an accelerating voltage of 3 kV.

Analysis of Molecular Weight Reduction of PHB Film
GPC (YL Chromass, Korea) was performed to determine the molecular weight and mass distribution of PHB.For GPC analysis, 1 g/l PHB pellets were emulsified in chloroform at 60°C for 2 h.Chloroform was evaporated in a fume hood until a plastic film formed.The PHB films were collected after incubation, washed with DW, and dried in an oven.For sample preparation, the PHB film was dissolved in chloroform at 60°C for 1 h.This solution was filtered through a 0.2-μm pore size syringe filter (Chromdisc, Korea) to separate the dissolved PHB from the remaining insoluble components.A high-performance liquid chromatography (HPLC) system consisting of a loop injector (Rheodyne 7725i), an isocratic pump with dual heads (YL9112), a column oven (YL9131), columns (Shodex, K-805, 8.0 I.D. × 300 mm, Shodex, K-804, 8.0 I.D. × 300 mm), and an refractive index (RI) detector (YL9170) was used for analysis.Sixty microliters of the solution without air bubbles were injected.Chloroform was used as the mobile phase at a flow rate of 1 ml/min and a temperature of 40°C.The data were analyzed using the YL-Clarity software for a single YL HPLC instrument (YL Chromass).Molecular masses were analyzed in relation to polystyrene standards ranging 5000-2,000,000 g/mol [21].

Isolation of B. infantis Strains
PHB-degrading bacteria were isolated from soil samples from landfills in Sejong, Korea, and rice fields in Suwon, Korea, where plastic waste was buried and generated.Soil samples were collected from a depth of 10 cm below the soil surface.After diluting the collected samples, various strains were isolated based on their morphological characteristics, such as colony size and color.A halo assay was performed to test the ability of the isolated strains; three strains showed the fastest degradation rate.By analyzing the 16S rRNA of the three strains that formed the halo zone, it was confirmed that all three strains were B. infantis with high similarity (homology 100, 99.9, and 100% for B. infantis PD1, PD2, and PD3, respectively), and were named B. infantis PD1, B. infantis PD2, and B. infantis PD3.They were deposited at the Biological Resources Center of the Korea Research Institute of Bioscience and Biotechnology under the accession numbers KCTC 19079P, KCTC 19080P, and KCTC 19081P, respectively.Interestingly, no halo zone was formed in TSB with a nitrogen source (1.7% tryptone and 0.3% soytone), but a distinct halo zone was formed in MM without a nitrogen source (Fig. 1A).To screen for PHBdegrading bacteria, strains are typically cultured on PHB-containing agar plates for 3-7 d, or more than 3 weeks [8,17,22].In this study, all three B. infantis strains isolated from soil formed a clear halo zone within 1 d, and the PHB double-layer plate was effective for the PHB halo assay.

Identification of phaZ and bdhA in B. infantis Using Universal Primers
PhaZ and BdhA are involved in PHB degradation pathway [15].PhaZ hydrolyzes PHB to 3-hydroxybutyrate, and BdhA converts 3-hydroxybutyrate to acetoacetate.These two important PHB-degrading enzymes in B. infantis PD1, PD2, and PD3, were identified using PCR with a Bacillus species-specific universal primer set and optimized PCR conditions.Primer sets used to identify the phaZ and bdhA genes of each enzyme were established by searching the nucleotide sequences of Bacillus species in the NCBI database.Primer sets were designed to amplify the conserved regions of 1.1 of 1.77 kb for phaZ and 280 of 780 bp for bdhA.The phaZ conserved region for amplification using PCR is relatively long; therefore, stable and reliable phaZ detection can be expected [23].The primers were designed by aligning the sequences of Bacillus species in the NCBI database using MEGA-X software (Figs.1B and 1C).Additionally, we developed a method to identify phaZ and bdhA at the gene level.The presence of phaZ and bdhA in all three strains was confirmed using electrophoresis (Fig. 1D).Sequencing revealed that the nucleotide sequences of each band obtained after electrophoresis in the three strains were consistent with those of phaZ and bdhA in B. infantis registered in the NCBI database.Therefore, B. infantis PD1, PD2, and PD3 are considered to have both types of genes encoding the key enzymes that degrade PHB.

Analysis of PHB Film Degradation in Liquid Medium
PHB is used as a carbon source for microorganism metabolism and is known to be degraded under conditions where nitrogen sources are limited.Therefore, TSB rich in nitrogen and MM without nitrogen were used to determine which medium was suitable for PHB degradation.TSB is the optimal medium for the growth of B. infantis.B. infantis PD1 and PD2 exhibited stable growth curves in TSB (Fig. 3A); however, their PHB film degradation rates were only 2.31% and 3.19%, respectively (Fig. 2A).B. infantis PD3 showed the highest optical  density (OD) 600 and PHB film degradation rate (6.39 %) in TSB.The same experiment was performed by limiting the nitrogen source in the medium compositions.Compared to TSB, the growth of B. infantis PD1, PD2, and PD3 decreased in MM without PHB film (Fig. 3B).The PHB film degradation rate was 4.25% for B. algicola SOL02, 30.29% for B. infantis PD1, 54.41% for B. infantis PD2, and 98.71% for B. infantis PD3 after 5 d (Fig. 2B).In B. infantis PD3, most of the PHB film was degraded within 5 d under a limited nitrogen source.In the case of B. algicola SOL02, which is known to degrade PHB, growth was not observed, which was expected because TSB is not an appropriate medium for the growth of B. algicola SOL02 [8].The growth of the three B. infantis PD1, PD2, and PD3 strains was reduced in MM because the nitrogen source required for bacterial growth was insufficient.However, the growth of all three B. infantis improved when the PHB film was present, indicating that the isolated and selected B. infantis can effectively degrade PHB under conditions containing various minerals using PHB as a carbon source for bacterial growth.In particular, B. infantis PD3 can be cultured in a limited nitrogen environment and has an excellent PHB degradation ability.Previous studies have shown that PHB degradation is induced and promoted under stressful conditions such as nitrogen limitation and poor nutrition [24,25].Consistent with previous reports, in the present study, PHB degradation was effectively induced by providing PHB as the sole carbon source and limiting nitrogen, which can stress the strains.Because these advantages are economically and environmentally suitable for the treatment of biodegradable plastic waste, an economical and eco-friendly approach can be expected using B. infantis PD3 in an industrial composting facility to reduce the cost and time required for processing biodegradation [26].

Analysis of Physical Properties of Degraded PHB Film
Changes in the surface morphology and molecular weight of PHB films were investigated after biodegradation by B. infantis PD3 in each medium.Before degradation, the initial PHB film exhibited a smooth surface with no fragments.The surface of the PHB film incubated in TSB for 5 d appeared to be slightly fragmented.The PHB films collected after biodegradation in MM for 5 d were highly fragmented into several small pieces (Fig. 4).Disappearance of the surface gloss of the film was also observed after PHB film degradation, indicating that the surface of the PHB film may have been eroded by PHB depolymerase [27].SEM images were analyzed to confirm the surface erosion of the PHB film collected after PHB film degradation.The PHB film surfaces were coated with platinum for electron scattering and observed at 1,500× and 10,000× magnification for each experiment (Fig. 5).The surface of the initial PHB film was smooth and exhibited no cracks.The surface became rough and cracked, resulting in unevenness.The surface of the PHB film in the MM was rougher than the surface of the PHB film in  the TSB, which was consistent with the result of PHB film degradation in liquid medium (Fig. 2).To further verify the changes in the physical properties of the degraded PHB films, their molecular weights were analyzed using GPC.As shown in Table 2, the average molecular weights of the degraded PHB film were number average (Mn) 37,661 and weight average (Mw) 169,538, which are significantly lower than those of the initial PHB film.When incubated without cells, these numbers changed slightly to Mn 117,859 and Mw 311,160.The polydispersity index (PDI; Mw/Mn) increased from 2.57 to 4.51, suggesting that the molecular structure of the PHB film changed and degraded into molecules of various sizes during degradation by B. infantis PD3.

Potential for Degradation of Other Bioplastics
A halo assay was performed to determine the potential of B. infantis PD3 to degrade other bioplastics, such as PBS and PBAT.Unlike PHB, PBS and PBAT are petroleum-based bioplastics.Bioplastics have physical properties that are easily compostable and biodegradable in natural environments [28].The halo assay was performed using B. infantis PD1, PD2, and PD3 in PBS and PBAT double-layer plates containing MM.As shown in Table 4, no halo zone was observed for 5 d in the PBS and PBAT double-layer plates for any of the three strains, indicating that B. infantis cannot degrade PBS and PBAT.Thus, we need to consider the possibility that PBS and PBAT require more time to degrade than PHB and that other enzymes are responsible for and specific to PBS and PBAT biodegradation [29,30].

Conclusion
PHB is a promising biodegradable plastic; however, effective degradation facilities are required for commercialization.For effective PHB degradation in soil and compost, we isolated three B. infantis strains with excellent PHB-degrading abilities from soil.To identify the presence of the PHB-degrading enzyme, Bacillus spp.universal primer sets were established, and the presence of phaZ and bdhA was confirmed using PCR.In addition, the PHB degradation ability improved when the nitrogen source was limited to the medium composition.B. infantis PD3 exhibited a high rate of PHB degradation (98.71%).In addition, the degraded PHB film had a decreased molecular weight and surface erosion.

Poly( 3 -
hydroxybutyrate) (PHB) is a biodegradable and biocompatible bioplastic.Effective PHB degradation in nutrient-poor environments is required for industrial and practical applications of PHB.To screen for PHB-degrading strains, PHB double-layer plates were prepared and three new Bacillus infantis species with PHB-degrading ability were isolated from the soil.In addition, phaZ and bdhA of all isolated B. infantis were confirmed using a Bacillus sp.universal primer set and established polymerase chain reaction conditions.To evaluate the effective PHB degradation ability under nutrient-deficient conditions, PHB film degradation was performed in mineral medium, resulting in a PHB degradation rate of 98.71% for B. infantis PD3, which was confirmed in 5 d.Physical changes in the degraded PHB films were analyzed.The decrease in molecular weight due to biodegradation was confirmed using gel permeation chromatography and surface erosion of the PHB film was observed using scanning electron microscopy.To the best of our knowledge, this is the first study on B. infantis showing its excellent PHB degradation ability and is expected to contribute to PHB commercialization and industrial composting.Keywords: Poly(3-hydroxybutyrate), biodegradation, Bacillus infantis, PHB depolymerase, phaZ, bdhA August 2023  Vol.33  No. 8

Fig. 3 .
Fig. 3. Cell growth curves of B. infantis by medium compositions with or without PHB film.Strains were cultured for 5 d in conditions with or without PHB.(A) TSB.(B) MM.Each bar represents the mean ± SD of three independent experiments.

Fig. 5 .
Fig. 5. SEM analysis of PHB film surfaces incubated with B. infantis PD3 in TSB and MM for 5 d.

Fig. 4 .
Fig. 4. PHB film following degradation for physical analysis.PHB film was recovered after 5 d of incubation, washed with DW, and dried.(A) Before degradation.(B) After degradation in TSB.(C) After degradation in MM.