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.

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.

Fructose-Based Production of Short-Chain-Length and Medium-Chain-Length Polyhydroxyalkanoate Copolymer by Arctic Pseudomonas sp. B14-6.

Arctic bacteria employ various mechanisms to survive harsh conditions, one of which is to accumulate carbon and energy inside the cell in the form of polyhydroxyalkanoate (PHA). Whole-genome sequencing of a new Arctic soil bacterium Pseudomonas sp. B14-6 revealed two PHA-production-related gene clusters containing four PHA synthase genes (phaC). Pseudomonas sp. B14-6 produced poly(6% 3-hydroxybutyrate-co-94% 3-hydroxyalkanoate) from various carbon sources, containing short-chain-length PHA (scl-PHA) and medium-chain-length PHA (mcl-PHA) composed of various monomers analyzed by GC-MS, such as 3-hydroxybutyrate, 3-hydroxyhexanoate, 3-hydroxyoctanoate, 3-hydroxydecanoate, 3-hydroxydodecenoic acid, 3-hydroxydodecanoic acid, and 3-hydroxytetradecanoic acid. By optimizing the PHA production media, we achieved 34.6% PHA content using 5% fructose, and 23.7% PHA content using 5% fructose syrup. Differential scanning calorimetry of the scl-co-mcl PHA determined a glass transition temperature (Tg) of 15.3 °C, melting temperature of 112.8 °C, crystallization temperature of 86.8 °C, and 3.82% crystallinity. In addition, gel permeation chromatography revealed a number average molecular weight of 3.6 × 104, weight average molecular weight of 9.1 × 104, and polydispersity index value of 2.5. Overall, the novel Pseudomonas sp. B14-6 produced a polymer with high medium-chain-length content, low Tg, and low crystallinity, indicating its potential use in medical applications.

Tung Oil-Based Production of High 3-Hydroxyhexanoate-Containing Terpolymer Poly(3-Hydroxybutyrate-co-3-Hydroxyvalerate-co-3-Hydroxyhexanoate) Using Engineered Ralstonia eutropha.

Polyhydroxyalkanoates (PHAs) are attractive new bioplastics for the replacement of plastics derived from fossil fuels. With their biodegradable properties, they have also recently been applied to the medical field. As poly(3-hydroxybutyrate) produced by wild-type Ralstonia eutropha has limitations with regard to its physical properties, it is advantageous to synthesize co- or terpolymers with medium-chain-length monomers. In this study, tung oil, which has antioxidant activity due to its 80% α-eleostearic acid content, was used as a carbon source and terpolymer P(53 mol% 3-hydroxybytyrate-co-2 mol% 3-hydroxyvalerate-co-45 mol% 3-hydroxyhexanoate) with a high proportion of 3-hydroxyhexanoate was produced in R. eutropha Re2133/pCB81. To avail the benefits of α-eleostearic acid in the tung oil-based medium, we performed partial harvesting of PHA by using a mild water wash to recover PHA and residual tung oil on the PHA film. This resulted in a film coated with residual tung oil, showing antioxidant activity. Here, we report the first application of tung oil as a substrate for PHA production, introducing a high proportion of hydroxyhexanoate monomer into the terpolymer. Additionally, the residual tung oil was used as an antioxidant coating, resulting in the production of bioactive PHA, expanding the applicability to the medical field.

Adsorptive removal of crude petroleum oil from water using floating pinewood biochar decorated with coconut oil-derived fatty acids.

The present investigation deals with the adsorptive removal of crude petroleum oil from the water surface using coconut oil-modified pinewood biochar. Biochar generated at higher pyrolysis temperature (700 °C) revealed higher fatty acid-binding efficiency responsible for the excellent hydrophobicity of the biochar. Fatty acids composition attached to the biochar produced at 700 °C was (mg g-1 BC) lauric acid (9.024), myristic acid (5.065), palmitic acid (2.769), capric acid (1.639), oleic acid (1.362), stearic acid (1.114), and linoleic acid (0.130). Simulation of the experimental adsorption data of pristine and modified pinewood biochar generated at 700 °C offered the best fit to pseudo-first-order kinetics (R2 > 0.97) and Langmuir isotherm model (R2 > 0.99) based on the highest regression coefficients. Consequently, the adsorption process was mainly driven by surface hydrophobic interactions including π-π electron-donor-acceptor between electron-rich (π-donor) polycyclic aromatic hydrocarbons from the crude oil and biochar (π-acceptor). A maximum adsorption capacity (Qmax) of 5.315 g g-1 was achieved by modified floating biochar within 60 min. Whereas the reusability testing revealed 49.39% and 51.40% was the adsorption efficiency of pristine and modified biochar at the fifth adsorption-desorption cycle.

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.

Increased Antibiotic Resistance of Methicillin-Resistant Staphylococcus aureus USA300 Δpsm Mutants and a Complementation Study of Δpsm Mutants Using Synthetic Phenol-Soluble Modulins.

Phenol-soluble modulins (PSMs) are responsible for regulating biofilm formation, persister cell formation, pmtR expression, host cell lysis, and anti-bacterial effects. To determine the effect of psm deletion on methicillin-resistant Staphylococcus aureus, we investigated psm deletion mutants including Δpsmα, Δpsmß, and Δpsmαß;. These mutants exhibited increased ß-lactam antibiotic resistance to ampicillin and oxacillin that was shown to be caused by increased Nacetylmannosamine kinase (nanK) mRNA expression, which regulates persister cell formation, leading to changes in the pattern of phospholipid fatty acids resulting in increased anteiso-C15:0, and increased membrane hydrophobicity with the deletion of PSMs. When synthetic PSMs were applied to Δpsmα and Δpsmß mutants, treatment of Δpsmα with PSMα1-4 and Δpsmß with PSMß1-2 restored the sensitivity to oxacillin and slightly reduced the biofilm formation. Addition of a single fragment showed that α1, α2, α3, and ß2 had an inhibiting effect on biofilms in Δpsmα; however, ß1 showed an enhancing effect on biofilms in Δpsmß. This study demonstrates a possible reason for the increased antibiotic resistance in psm mutants and the effect of PSMs on biofilm formation.

Revealing of sugar utilization systems in Halomonas sp. YLGW01 and application for poly(3-hydroxybutyrate) production with low-cost medium and easy recovery.

Poly(3-hydroxybutyrate) (PHB) is a common polyhydroxyalkanoate (PHA) with potential as an alternative for petroleum-based plastics. Previously, we reported a new strain, Halomonas sp. YLGW01, which hyperproduces PHB with 94% yield using fructose. In this study, we examined the PHB production machinery of Halomonas sp. YLGW01 in more detail by deep-genome sequencing, which revealed a 3,453,067-bp genome with 65.1% guanine-cytosine content and 3054 genes. We found two acetyl-CoA acetyltransferases (Acetoacetyl-CoA thiolase, PhaA), one acetoacetyl-CoA reductase (PhaB), two PHB synthases (PhaC1, PhaC2), PHB depolymerase (PhaZ), and Enoyl-CoA hydratase (PhaJ) in the genome, along with two fructose kinases and fructose transporter systems, including the phosphotransferase system (PTS) and ATP-binding transport genes. We then examined the PHB production by Halomonas sp. YLGW01 using high-fructose corn syrup (HFCS) containing fructose, glucose, and sucrose in sea water medium, resulting in 7.95 ± 0.11 g/L PHB (content, 67.39 ± 0.34%). PHB was recovered from Halomonas sp. YLGW01 using different detergents; the use of Tween 20 and SDS yielded micro-sized granules with high purity. Overall, these results reveal the distribution of PHB synthetic genes and the sugar utilization system in Halomonas sp. YLGW01 and suggest a possible method for PHB recovery.

NaCl Concentration-Dependent Aminoglycoside Resistance of Halomonas socia CKY01 and Identification of Related Genes.

Among various species of marine bacteria, those belonging to the genus Halomonas have several promising applications and have been studied well. However, not much information has been available on their antibiotic resistance. In our efforts to learn about the antibiotic resistance of strain Halomonas socia CKY01, which showed production of various hydrolases and growth promotion by osmolytes in previous study, we found that it exhibited resistance to multiple antibiotics including kanamycin, ampicillin, oxacillin, carbenicillin, gentamicin, apramycin, tetracycline, and spectinomycin. However, the H. socia CKY01 resistance pattern to kanamycin, gentamicin, apramycin, tetracycline, and spectinomycin differed in the presence of 10% NaCl and 1% NaCl in the culture medium. To determine the mechanism underlying this NaCl concentration-dependent antibiotic resistance, we compared four aminoglycoside resistance genes under different salt conditions while also performing time-dependent reverse transcription PCR. We found that the aph2 gene encoding aminoglycoside phosphotransferase showed increased expression under the 10% rather than 1% NaCl conditions. When these genes were overexpressed in an Escherichia coli strain, pETDuet-1::aph2 showed a smaller inhibition zone in the presence of kanamycin, gentamicin, and apramycin than the respective control, suggesting aph2 was involved in aminoglycoside resistance. Our results demonstrated a more direct link between NaCl and aminoglycoside resistance exhibited by the H. socia CKY01 strain.

Effects of a Δ-9-fatty acid desaturase and a cyclopropane-fatty acid synthase from the novel psychrophile Pseudomonas sp. B14-6 on bacterial membrane properties.

Psychrophilic bacteria, living at low and mild temperatures, can contribute significantly to our understanding of microbial responses to temperature, markedly occurring in the bacterial membrane. Here, a newly isolated strain, Pseudomonas sp. B14-6, was found to dynamically change its unsaturated fatty acid and cyclic fatty acid content depending on temperature which was revealed by phospholipid fatty acid (PLFA) analysis. Genome sequencing yielded the sequences of the genes Δ-9-fatty acid desaturase (desA) and cyclopropane-fatty acid-acyl-phospholipid synthase (cfa). Overexpression of desA in Escherichia coli led to an increase in the levels of unsaturated fatty acids, resulting in decreased membrane hydrophobicity and increased fluidity. Cfa proteins from different species were all found to promote bacterial growth, despite their sequence diversity. In conclusion, PLFA analysis and genome sequencing unraveled the temperature-related behavior of Pseudomonas sp. B14-6 and the functions of two membrane-related enzymes. Our results shed new light on temperature-dependent microbial behaviors and might allow to predict the consequences of global warming on microbial communities.

Combination Therapy Using Low-Concentration Oxacillin with Palmitic Acid and Span85 to Control Clinical Methicillin-Resistant Staphylococcus aureus.

The overuse of antibiotics has led to the emergence of multidrug-resistant bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA). MRSA is difficult to kill with a single antibiotic because it has evolved to be resistant to various antibiotics by increasing the PBP2a (mecA) expression level, building up biofilm, introducing SCCmec for multidrug resistance, and changing its membrane properties. Therefore, to overcome antibiotic resistance and decrease possible genetic mutations that can lead to the acquisition of higher antibiotic resistance, drug combination therapy was applied based on previous results indicating that MRSA shows increased susceptibility to free fatty acids and surfactants. The optimal ratio of three components and the synergistic effects of possible combinations were investigated. The combinations were directly applied to clinically isolated strains, and the combination containing 15 µg/mL of oxacillin was able to control SCCmec type III and IV isolates having an oxacillin minimum inhibitory concentration (MIC) up to 1024 µg/mL; moreover, the combination with a slightly increased oxacillin concentration was able to kill SCCmec type II. Phospholipid analysis revealed that clinical strains with higher resistance contained a high portion of 12-methyltetradecanoic acid (anteiso-C15:0) and 14-methylhexadecanoic acid (anteiso-C17:0), although individual strains showed different patterns. In summary, we showed that combinatorial therapy with a low concentration of oxacillin controlled different laboratory and highly diversified clinical MRSA strains.

Enhancement of pipecolic acid production by the expression of multiple lysine cyclodeaminase in the Escherichia coli whole-cell system.

Pipecolic acid, a non-proteinogenic amino acid, is a metabolite in lysine metabolism and a key chiral precursor in local anesthesia and macrolide antibiotics. To replace the environmentally unfriendly chemical production or preparation procedure of pipecolic acid, many biological synthetic routes have been studied for a long time. Among them, synthesis by lysine cyclodeaminase (LCD), encoded by pipA, has several advantages, including stability of enzyme activity and NAD+ self-regeneration. Thus, we selected this enzyme for pipecolic acid biosynthesis in a whole-cell bioconversion. To construct a robust pipecolic acid production system, we investigated important conditions including expression vector, strain, culture conditions, and other reaction parameters. The most important factor was the introduction of multiple pipA genes into the whole-cell system. As a result, we produced 724 mM pipecolic acid (72.4 % conversion), and the productivity was 0.78 g/L/h from 1 M l-lysine after 5 days. This is the highest production reported to date.

Enhanced isobutanol production by co-production of polyhydroxybutyrate and cofactor engineering.

Once cells have been used to produce biochemicals, there are only a few effective ways to utilize the residual cell mass, even though the utilization of leftover cells would aid in decreasing production costs. Here, a polyhydroxybutyrate (PHB) and isobutanol co-production system was designed to address this challenge. The addition of the PHB operon into Escherichia coli conferred a metabolic advantage for alcohol production, generating 1.14-fold more isobutanol. Furthermore, following nitrogen source optimization and cofactor engineering, the engineered E. coli strain produced 2-fold more isobutanol and 0.25 g/L PHB. Moreover, E. coli cells showed higher tolerance to isobutanol with the overexpression of PHB biosynthesis genes. This co-production system resulted in an increased biomass, higher glucose utilization, and lower acetate maintenance, leading to higher productivity regarding PHB and isobutanol yield. Thus, this novel system is applicable to future fermentation studies for the co-production of PHB and isobutanol.

Fructose based hyper production of poly-3-hydroxybutyrate from Halomonas sp. YLGW01 and impact of carbon sources on bacteria morphologies.

Polyhydroxyalkanoates (PHA), such as poly (3-hydroxybutyrate) (PHB), have emerged as potential alternatives to petroleum-based plastics and can be produced through the appropriate selection of marine bacteria that are already adapted to high salt and low temperature conditions without the requirement of antibiotic treatment. The present study, thus, aimed to screen and characterize thirteen PHA-producing microbial strains isolated from the Gwangalli beach in Busan, South Korea. Among them, Halomonas sp. YLGW01 produced the highest amount of PHB (94.6 ± 1.8% (w/w)) using fructose. Interestingly Halomonas sp. YLGW01 showed increase in cell size (8.39 ± 3.63 µm) with fructose as carbon source as compared to glucose (2.34 ± 0.44 µm). Fructose syrup was investigated as carbon source under unsterilized conditions and 95.26 ± 1.78% of PHB was produced. Overall, this strain showed the highest PHB contents in halotolerant bacteria.