Revealing the key gene involved in bioplastic degradation from superior bioplastic degrader Bacillus sp. JY35.

The use of bioplastics, which can alleviate environmental pollution caused by non-degradable bioplastics, has received attention. As there are many types of bioplastics, method that can treat them simultaneously is important. Therefore, Bacillus sp. JY35 which can degrade different types of bioplastics, was screened in previous study. Most types of bioplastics, such as polyhydroxybutyrate (PHB), (P(3HB-co-4HB)), poly(butylene adipate-co-terephthalate) (PBAT), polybutylene succinate (PBS), and polycaprolactone (PCL), can be degraded by esterase family enzymes. To identify the genes that are involved in bioplastic degradation, analysis with whole-genome sequencing was performed. Among the many esterase enzymes, three carboxylesterase and one triacylglycerol lipase were identified and selected based on previous studies. Esterase activity using p-nitrophenyl substrates was measured, and the supernatant of JY35_02679 showed strong emulsion clarification activity compared with others. In addition, when recombinant E. coli was applied to the clear zone test, only the JY35_02679 gene showed activity in the clear zone test with bioplastic containing solid cultures. Further quantitative analysis showed 100 % PCL degradation at 7 days and 45.7 % PBS degradation at 10 days. We identified a gene encoding a bioplastic-degrading enzyme in Bacillus sp. JY35 and successfully expressed the gene in heterologous E. coli, which secreted esterases with broad specificity.

Acceleration of Polybutylene Succinate Biodegradation by Terribacillus sp. JY49 Isolated from a Marine Environment.

Polybutylene succinate (PBS) is a bioplastic substitute for synthetic plastics that are made from petroleum-based products such as polyethylene and polypropylene. However, the biodegradation rate of PBS is still low and similar to that of polylactic acid (PLA). Moreover, our knowledge about degrader species is limited to a few fungi and mixed consortia. Here, to identify a bacterial degrader to accelerate PBS degradation, we screened and isolated Terribacillus sp. JY49, which showed significant degradability. In order to optimize solid and liquid culture conditions, the effect of factors such as temperature, additional carbon sources, and salt concentrations on degradation was confirmed. We observed a degradation yield of 22.3% after 7 days when adding 1% of glucose. Additionally, NaCl was added to liquid media, and degradation yield was decreased but PBS films were broken into pieces. Comparing the degree of PBS degradation during 10 days, the degradation yield was 31.4% after 10 days at 30 °C. Alteration of physical properties of films was analyzed by using scanning electron microscopy (SEM), gel permeation chromatography (GPC), and Fourier transform infrared (FT-IR). In addition, Terribacillus sp. JY49 showed clear zones on poly(butylene adipate-co-terephthalate) (PBAT), polycaprolactone (PCL), and copolymers such as P(3HB-co-3HV) and P(3HV-co-4HB), exhibiting a broad spectrum of degradation activities on bioplastics. However, there was no significant difference in absorbance when esterase activity was examined for different types of bioplastics. Overall, Terribacillus sp. JY49 is a potential bacterial strain that can degrade PBS and other bioplastics, and this is the first report of Terribacillus sp. as a bioplastic degrader.

Finding a Benign Plasticizer to Enhance the Microbial Degradation of Polyhydroxybutyrate (PHB) Evaluated by PHB Degrader Microbulbifer sp. SOL66.

As a biodegradable plastic, polyhydroxybutyrate (PHB) has relatively poor mechanical properties, preventing its wider use. Various plasticizers have been studied to improve the mechanical properties of PHB; however, due to the slow degradation speed in the soil environment and lack of evaluation methods, studies on the degradation of PHB with plasticizers are rarely reported. In this study, by applying Microbulbifer sp. SOL66, which is able to degrade PHB very quickly, a benign plasticizer was evaluated with good properties and good degradability, not inhibiting microbial activities. Eight different plasticizers were applied with PHB and Microbulbifer sp. SOL66, PHB film containing 10% and 20% tributyl citrate showed significant biodegradability of PHB. It was confirmed that tributyl citrate could increase the speed of PHB degradation by Microbulbifer sp. SOL66 by 88% at 1 day, although the degree of degradation was similar after 3 days with and without tributyl citrate. By the analysis of microbial degradation, physical, chemical, and mechanical properties, tributyl citrate was shown not only to improve physical, chemical, and mechanical properties but also the speed of microbial degradation.

Simultaneous monitoring of each component on degradation of blended bioplastic using gas chromatography-mass spectrometry.

The increasing interest in bioplastics, with regard to future environmental issues, has rendered research on bioplastic biodegradation highly important. However, only a few tools directly monitor the degradation of bioplastics without measuring the levels of gaseous products, such as carbon dioxide. Classical nonquantitative methods, such as clear zone tests on solid plates, and less-sensitive weight-loss experiments in liquid media measured using a precision scale, are still employed to screen the microbial players associated with bioplastic degradation and monitor the biodegradation rates. However, the simultaneous monitoring of the degradation of each component of blended bioplastics has not been previously reported. In the present study, to provide information regarding the degradation rates and compositional changes of different bioplastics in a blend in a time-dependent manner, we simultaneously monitored and quantified the degradation of four bioplastics, polyhydroxybutyrate (PHB), polybutylene succinate (PBS), polycaprolactone (PCL), and poly(butylene adipate-co-terephthalate) (PBAT), by Bacillus sp. JY36 using gas chromatography-mass spectrometry (GC-MS) analysis after fatty acid methyl ester (FAME) derivatization. Our results demonstrate the feasibility of using the GC-MS-based method described here to obtain comprehensive data regarding blended bioplastics and their degradation. Moreover, our findings indicate that this method may support classical analytic tools for assessing bioplastic biodegradation.

Novel Poly(butylene adipate-co-terephthalate)-degrading Bacillus sp. JY35 from wastewater sludge and its broad degradation of various bioplastics.

Poly(butylene adipate-co-terephthalate) (PBAT), a bioplastic consisting of aliphatic hydrocarbons and aromatic hydrocarbons, was developed to overcome the shortcomings of aliphatic and aromatic polyesters. Many studies report the use of PBAT as a blending material for improving properties of other bioplastics. However, there are few studies on microorganisms that degrade PBAT. We found six kinds of PBAT-degrading microorganisms from various soils. Among these, Bacillus sp. JY35 showed superior PBAT degradability and robustness to temperature. We monitored the degradation of PBAT films by Bacillus sp. JY35 using scanning electron microscopy, field emission scanning electron microscopy, Fourier-transform infrared spectroscopy, and gel permeation chromatography. GC-MS was used to measure the PBAT film degradation rate at different temperatures and with additional NaCl and carbon sources. Certain additional carbon sources improve the growth of Bacillus sp. JY35. However, this did not increase PBAT film degradation. Time-dependent PBAT film degradation rates were measured during three weeks of cultivation, after which the strain achieved almost 50% degradation. Additionally, various bioplastics were applied to solid cultures to confirm the biodegradation range of Bacillus sp. JY35, which can degrade not only PBAT but also PBS, PCL, PLA, PHB, P(3HB-co-4HB), P(3HB-co-3HV), P(3HB-co-3HHx), and P(3HB-co-3HV-co-3HHx), suggesting its usability as a superior bioplastic degrader.

Adsorptive removal of synthetic plastic components bisphenol-A and solvent black-3 dye from single and binary solutions using pristine pinecone biochar.

The existing study deals with adsorptive removal of the endocrine-disrupting chemical bisphenol-A and toxic azo dye solvent black-3 from single and binary solutions. These two chemicals are commonly used as an additive in the synthetic plastic industries. Among the tested twenty pristine and modified biochars, the pristine pinecone biochar produced at 750 °C revealed greater bisphenol-A removal. Simulation of the experimental data obtained for bisphenol-A and dye removal from the single-component solution offered a best-fit to Elovich (R2 > 0.98) and pseudo-second-order (R2 > 0.99) kinetic models, respectively. Whereas for the bisphenol-A + dye removal from binary solution, the values for bisphenol-A adsorption were best suited to Elovich (R2 > 0.98), while pseudo-second-order (R2 > 0.99) for dye removal. Similarly, the two-compartment model also demonstrated better values (R2 > 0.92) for bisphenol-A and dye removal from single and binary solutions with greater Ffast values (except for bisphenol-A in binary solution). The Langmuir isotherm model demonstrated the highest regression coefficient values (R2 > 0.99) for bisphenol-A and dye removal with the highest adsorption capacity of 38.387 mg g-1 and 346.856 mg g-1, correspondingly. Besides, the co-existence of humic acid revealed a positive impact on bisphenol-A removal, while the dye removal rate was slightly hindered in presence of humic acid. The absorption process showed monolayer coverage of biochar surface with contaminants using a chemisorption mechanism with fast reactions between functional groups on the adsorbate and adsorbent. Whereas the adsorption mechanism was primarily controlled by hydrogen bonding, hydrophobic and π-π electron-donor-acceptor interactions as confirmed by FTIR, XPS, and pH investigations.

Novel Polyhydroxybutyrate-Degrading Activity of the Microbulbifer Genus as Confirmed by Microbulbifer sp. SOL03 from the Marine Environment.

Ever since bioplastics were globally introduced to a wide range of industries, the disposal of used products made with bioplastics has become an issue inseparable from their application. Unlike petroleum-based plastics, bioplastics can be completely decomposed into water and carbon dioxide by microorganisms in a relatively short time, which is an advantage. However, there is little information on the specific degraders and accelerating factors for biodegradation. To elucidate a new strain for biodegrading poly-3-hydroxybutyrate (PHB), we screened out one PHB-degrading bacterium, Microbulbifer sp. SOL03, which is the first reported strain from the Microbulbifer genus to show PHB degradation activity, although Microbulbifer species are known to be complex carbohydrate degraders found in high-salt environments. In this study, we evaluated its biodegradability using solid- and liquid-based methods in addition to examining the changes in physical properties throughout the biodegradation process. Furthermore, we established the optimal conditions for biodegradation with respect to temperature, salt concentration, and additional carbon and nitrogen sources; accordingly, a temperature of 37°C with the addition of 3% NaCl without additional carbon sources, was determined to be optimal. In summary, we found that Microbulbifer sp. SOL03 showed a PHB degradation yield of almost 97% after 10 days. To the best of our knowledge, this is the first study to investigate the potent bioplastic degradation activity of Microbulbifer sp., and we believe that it can contribute to the development of bioplastics from application to disposal.

Isolation of Microbulbifer sp. SOL66 with High Polyhydroxyalkanoate-Degrading Activity from the Marine Environment.

Having the advantage of eco-friendly decomposition, bioplastics could be used to replace petroleum-based plastics. In particular, poly(3-hydroxybutyrate) (PHB) is one of the most commercialized bioplastics, however, necessitating the introduction of PHB-degrading bacteria for its effective disposal. In this study, Microbulbifer sp. SOL66 (94.18% 16S rRNA with similarity to Microbulbifer hydrolyticus) demonstrated the highest degradation activity among five newly screened Microbulbifer genus strains. Microbulbifer sp. SOL66 showed a rapid degradation yield, reaching 98% in 4 days, as monitored by laboratory scale, gas chromatography-mass spectrometry, scanning electron microscopy, gel permeation chromatography, and Fourier transform infrared spectroscopy. The PHB film was completely degraded within 7 days at 37 °C in the presence of 3% NaCl. When 1% xylose and 0.4% ammonium sulfate were added, the degradation activity increased by 17% and 24%, respectively. In addition, this strain showed biodegradability on pellets of poly(3-hydroxybutyrate-co-4-hydroxybutyrate), as confirmed by weight loss and physical property changes. We confirmed that Microbulbifer sp. SOL66 has a great ability to degrade PHB, and has rarely been reported to date.

Novel phasins from the Arctic Pseudomonas sp. B14-6 enhance the production of polyhydroxybutyrate and increase inhibitor tolerance.

Phasin (PhaP), one of the polyhydroxyalkanoate granule-associated protein, enhances cell growth and polyhydroxybutyrate (PHB) biosynthesis by regulating the number and size of PHB granules. However, few studies have applied phasins to various PHB production conditions. In this study, we identified novel phasin genes from the genomic data of Arctic soil bacterium Pseudomonas sp. B14-6 and determined the role of phaP1Ps under different PHB production conditions. Transmission electron microscopy and gel permeation chromatography revealed small PHB granules with high-molecular weight, while differential scanning calorimetry showed that the extracted PHB films had similar thermal properties. The phasin protein derived from Pseudomonas sp. B14-6 revealed higher PHB production and exhibited higher tolerance to several lignocellulosic biosugar-based inhibitors than the phasin protein of Ralstonia eutropha H16 in a recombinant Escherichia coli strain. The increased tolerance to propionate, temperature, and other inhibitors was attributed to the introduction of phaP1Ps, which increased PHB production from lignocellulosic hydrolysate (2.39-fold) in the phaP1Ps strain. However, a combination of phasin proteins isolated from two different sources did not increase PHB production. These findings suggest that phasin could serve as a powerful means to increase robustness and PHB production in heterologous strains.

Comparative Study of the Difference in Behavior of the Accessory Gene Regulator (Agr) in USA300 and USA400 Community-Associated Methicillin-Resistant Staphylococcus aureus (CA-MRSA).

Community-associated Methicillin-Resistant Staphylococcus aureus (CA-MRSA) is notorious as a leading cause of soft tissue infections. Despite several studies on the Agr regulator, the mechanisms of action of Agr on the virulence factors in different strains are still unknown. To reveal the role of Agr in different CA-MRSA, we investigated the LACΔagr mutant and the MW2Δagr mutant by comparing LAC (USA300), MW2 (USA400), and Δagr mutants. The changes of Δagr mutants in sensitivity to oxacillin and several virulence factors such as biofilm formation, pigmentation, motility, and membrane properties were monitored. LACΔagr and MW2Δagr mutants showed different oxacillin sensitivity and biofilm formation compared to the LAC and MW2 strains. Regardless of the strain, the motility was reduced in Δagr mutants. And there was an increase in the long chain fatty acid in phospholipid fatty acid composition of Δagr mutants. Other properties such as biofilm formation, pigmentation, motility, and membrane properties were different in both Δagr mutants. The Agr regulator may have a common role like the control of motility and straindependent roles such as antibiotic resistance, biofilm formation, change of membrane, and pigment production. It does not seem easy to control all MRSA by targeting the Agr regulator only as it showed strain-dependent behaviors.

Polyhydroxyalkanoates (PHAs) degradation by the newly isolated marine Bacillus sp. JY14.

Polyhydroxyalkanoates (PHAs) are bioplastic substitutes for petroleum-derived plastics that may help to reduce the increasing environmental impact of plastic pollution. Among them, polyhydroxybutyrate (PHB) is a promising biopolymer, incentivizing many researchers to search for PHB-producing and PHB-degrading bacteria for improved PHB utilization. Many novel PHB-producing microorganisms have been discovered; however, relatively few PHB-degrading bacteria have been identified. Six PHB-degrading bacteria were found in marine soil and investigated their PHB-degrading abilities under various temperature and salinity conditions using solid-media based culture. Finally, thermotolerant and halotolerant PHB-degrader Bacillus sp. JY14 was selected. PHB degradation was confirmed by monitoring changes in the physical and chemical properties of PHB films incubated with Bacillus sp. JY14 using scanning electron microscopy, Fourier-transform infrared spectroscopy, and gel permeation chromatography. Further, PHB degradation ability of Bacillus sp. JY14 was measured in liquid culture by gas chromatography. After 14 days of cultivation with PHB film, Bacillus sp. JY14 achieved approximately 98% PHB degradation. Applying various bioplastics to assess the bacteria's biodegradation capabilities, the result showed that Bacillus sp. JY14 could also degrade P(3HB-co-4HB) and P(3HB-co-3HV). Overall, this study identified a thermotolerant and halotolerant bacteria capable of PHB degradation under solid and liquid conditions. These results suggest that this bacteria could be utilized to degrade various PHAs.

Engineering of Shewanella marisflavi BBL25 for biomass-based polyhydroxybutyrate production and evaluation of its performance in electricity production.

Polyhydroxybutyrate (PHB) is a biodegradable plastic with physical properties similar to petrochemically derived plastics. Here, Shewanella marisflavi BBL25 was engineered by inserting the pLW487 vector containing polyhydroxyalkanoates synthesis genes from Ralstonia eutropha H16. Under optimal conditions, the engineered S. marisflavi BBL25 produced 1.99 ± 0.05 g/L PHB from galactose. The strain showed high tolerance to various inhibitors and could utilize lignocellulosic biomass for PHB production. When barley straw hydrolysates were used as a carbon source, PHB production was 3.27 ± 0.19 g/L. In addition, PHB production under the microbial fuel cell system was performed to confirm electricity coproduction. The maximum electricity current output density was 1.71 mA/cm2, and dry cell weight (DCW) and PHB production were 11.4 g/L and 6.31 g/L, respectively. Our results demonstrated PHB production using various lignocellulosic biomass and the feasibility of PHB and electricity production, simultaneously, and it is the first example of PHB production in engineered Shewanella.

Screening of the strictly xylose-utilizing Bacillus sp. SM01 for polyhydroxybutyrate and its co-culture with Cupriavidus necator NCIMB 11599 for enhanced production of PHB.

Polyhydroxybutyrate (PHB) is a biodegradable plastic that can be used as an alternative to petrochemical-based plastics. PHB is produced by various microorganisms such as Ralstonia, Halomonas, and Bacillus species. However, there are very few strains that produce PHB using xylose, an abundant and inexpensive carbon source. In this study, ten xylose-utilizing PHB producers isolated from South Korean marine environments were screened and characterized. Among these isolates, Bacillus sp. SM01, a newly identified strain, produced the highest amount of PHB using xylose. Under optimal conditions, the maximum dry cell weight (DCW) was 3.41 ± 0.09 g/L, with 62% PHB content, and Bacillus sp. SM01 showed Poly (3-hydroxybutyrate-co-3-hydroxyvalerate) copolymer production with propionate; however, the growth of Bacillus sp. SM01 was greatly inhibited by the presence of glucose. Co-culturing Bacillus sp. SM01 with Cupriavidus necator NCIMB 11599 resulted in increased DCW, PHB production, and utilization of glucose and xylose, the main sugar of lignocellulosic biomass, compared with the monoculture. Our results indicated that this co-culture system can be used to increase PHB production and overcome the limitation of sugar consumption associated with Bacillus sp. SM01 and C. necator.

Improvement of polyhydroxybutyrate (PHB) plate-based screening method for PHB degrading bacteria using cell-grown amorphous PHB and recovered by sodium dodecyl sulfate (SDS).

Poly(3-hydroxybutyrate) (PHB) is a biobased and biodegradable plastic. Considering the environmental issues of petroleum-based plastics, PHB is promising as it can be degraded in a relatively short time by bacteria to water and carbon dioxide. Substantial efforts have been made to identify PHB-degrading bacteria. To identify PHB-degrading bacteria, solid-based growth or clear zone assays using PHB as the sole carbon source are the easiest methods; however, PHB is difficult to dissolve and distribute evenly, and bacteria grow slowly on PHB plates. Here, we suggest an improved PHB plate assay using cell-grown PHB produced by Halomonas sp. and recovered by sodium dodecyl sulfate (SDS). Preparation using SDS resulted in evenly distributed PHB plates that could be used for sensitive depolymerase activity screening in less time compared with solvent-melted pellet or cell-grown PHB. With this method, we identified 15 new strains. One strain, Cutibacterium sp. SOL05 (98.4% 16S rRNA similarity to Cutibacterium acne), showed high PHB depolymerase activity in solid and liquid conditions. PHB degradation was confirmed by clear zone size, liquid culture, scanning electron microscopy, and Fourier-transform infrared spectroscopy. The results indicate this method can be used to easily identify PHB-degrading bacteria from various sources to strengthen the benefits of bioplastics.

Improvement of cadaverine production in whole cell system with baker's yeast for cofactor regeneration.

Cadaverine, 1,5-diaminopentane, is one of the most promising chemicals for biobased-polyamide production and it has been successfully produced up to molar concentration. Pyridoxal 5'-phosphate (PLP) is a critical cofactor for inducible lysine decarboxylase (CadA) and is required up to micromolar concentration level. Previously the regeneration of PLP in cadaverine bioconversion has been studied and salvage pathway pyridoxal kinase (PdxY) was successfully introduced; however, this system also required a continuous supply of adenosine 5'-triphosphate (ATP) for PLP regeneration from pyridoxal (PL) which add in cost. Herein, to improve the process further a method of ATP regeneration was established by applying baker's yeast with jhAY strain harboring CadA and PdxY, and demonstrated that providing a moderate amount of adenosine 5'-triphosphate (ATP) with the simple addition of baker's yeast could increase cadaverine production dramatically. After optimization of reaction conditions, such as PL, adenosine 5'-diphosphate, MgCl2, and phosphate buffer, we able to achieve high production (1740 mM, 87% yield) from 2 M L-lysine. Moreover, this approach could give averaged 80.4% of cadaverine yield after three times reactions with baker's yeast and jhAY strain. It is expected that baker's yeast could be applied to other reactions requiring an ATP regeneration system.