Yangia sp. CCB-MM3 was one of several halophilic bacteria isolated from soil sediment in the estuarine Matang Mangrove, Malaysia. So far, no member from the genus Yangia, a member of the Rhodobacteraceae family, has been reported sequenced. In the current study, we present the first complete genome sequence of Yangia sp. strain CCB-MM3. The genome includes two chromosomes and five plasmids with a total length of 5,522,061 bp and an average GC content of 65%. Since a different strain of Yangia sp. (ND199) was reported to produce a polyhydroxyalkanoate copolymer, the ability for this production was tested in vitro and confirmed for strain CCB-MM3. Analysis of its genome sequence confirmed presence of a pathway for production of propionyl-CoA and gene cluster for PHA production in the sequenced strain. The genome sequence described will be a useful resource for understanding the physiology and metabolic potential of Yangia as well as for comparative genomic analysis with other Rhodobacteraceae.
Long carbon chain alkanediols are used in the production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)], however these substrates possess high toxicity towards bacterial cells. This study demonstrated the effective utilisation of a long carbon chain alkanediol, namely 1,8-octanediol, to enhance the yield and production of a copolymer with a high molecular weight of over 1000 kDa, which is desirable for novel applications in medical and biopharmaceuticals. The increased PHA content (47-61 wt%) and concentration (1.7-4.5 g/L) was achieved by additional feeding of a combination of C4 substrates at C/N 10, with 1,8-octanediol + γ-butyrolactone producing P(3HB-co-22 mol% 4HB) with a high molecular weight (1060 kDa) and elongation at break of 970%. The DO-stat feeding strategy of C/N 10 has shown an increment of PHA concentration for both carbon combination, 0.45-4.27 g/L and 0.32-3.36 g/L for 1,8-octanediol + sodium 4-hydroxybutyrate (4HB-Na) and 1,8-octanediol + γ-butyrolactone, but with a slight reduction on molecular weight and mechanical strength. Nonetheless, further study revealed that a nitrogen-absence feeding strategy could retain the high molecular weight and elongation at break of the copolymer, and simultaneously improving the overall P(3HB-co-4HB) production.
This study reports an efficient fed-batch strategy to improve poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-4-hydroxybutyrate) [P(3HB-co-3HV-co-4HB)] terpolymer production by Cupriavidus sp. USMAA2-4 with enhanced mechanical properties in bioreactor. The cultivations have been performed by combining oleic acid with γ-butyrolactone at different concentration ratios with 1-pentanol at a fixed concentration. The batch and fed-batch fermentations have resulted in P(3HB-co-3HV-co-4HB) with compositions of 9-35 mol% 3HV and 4-24 mol% 4HB monomers. The DO-stat fed-batch fermentation strategies have significantly improved the production with a maximum 4.4-fold increment of cell dry weight (CDW). Besides, appropriate feeding of the substrates has resulted in an increment of terpolymer productivity from 0.086-0.347 g/L/h, with a significantly shortened cultivation time. The bacterial growth and terpolymer formation have been found to be affected by the concentration of carbon sources supplied. Characterization of P(3HB-co-3HV-co-4HB) has demonstrated that incorporation of 3HV and 4HB monomer has significantly improved the physical and thermodynamic properties of the polymers, by reducing the polymer's crystallinity. The tensile strength, Young's modulus of the terpolymer has been discovered to increase with the increase of M w. The fed-batch fermentation strategies employed in this study have resulted in terpolymers with a range of flexible materials having improved tensile strength and Young's modulus as compared to the terpolymer produced from batch fermentation. Possession of lower melting temperature indicates an enhanced thermal stability which broadens the polymer processing window.
Cupriavidus sp. USMAA1020, USMAA2-4, and USMAHM13 are capable of producing polyhydroxyalkanoate (PHA). This biopolymer is an alternative solution to synthetic plastics, whereby polyhydroxyalkanoate synthase is the key enzyme involved in PHA biosynthesis. Here, we report the complete genomes of three Cupriavidus sp. strains: USMAA1020, USMAA2-4, and USMAHM13.
Polyhydroxyalkanoate (PHA) is a biogenic polymer that has the potential to substitute synthetic plastic in numerous applications. This is due to its unique attribute of being a biodegradable and biocompatible thermoplastic, achievable through microbial fermentation from a broad utilizable range of renewable resources. Among all the PHAs discovered, poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] stands out as a next generation healthcare biomaterial for having high biopharmaceutical and medical value since it is highly compatible to mammalian tissue. This review provides a critical assessment and complete overview of the development and trend of P(3HB-co-4HB) research over the last few decades, highlighting aspects from the microbial strain discovery to metabolic engineering and bioprocess cultivation strategies. The article also outlines the relevance of P(3HB-co-4HB) as a material for high value-added products in numerous healthcare-related applications.
Separation of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] from bacterial cell matter is a critical step in the downstream process with respect to material quality and eco-balance as P(3HB-co-4HB) is widely used for biomedical applications. Therefore, an efficient and eco-based extraction of P(3HB-co-4HB) using a combination of NaOH and Lysol in digesting the non-polymeric cell material (NPCM) digestion is developed. The NaOH and Lysol show synergistic influence on the copolymer extraction at a high purity and recovery of 97 and 98 wt% respectively. The optimized cell digestion method was found applicable to a vast batch of cells containing copolymers from various 4HB monomer compositions. At the largest extraction volume of 100 L, P(3HB-co-4HB) with a purity of 89 wt% was extracted with a maximum recovery of 90 wt%. The method developed has also eliminated the cell pretreatment step. The extraction method developed in this research has not only produced an economic and efficient copolymer recovery but has also retained the copolymer quality, in term of its molecular weight and thermal properties. It demonstrates a practical and promising downstream processing method in recovering the copolymer effectively from the bacterial biomass.
Hahella sp. strain CCB-MM4 is a halophilic bacterium isolated from estuarine mangrove sediment. The genome sequence of Hahella sp. CCB-MM4 provides insights into exopolysaccharide biosynthesis and the lifestyle of the bacterium thriving in a saline mangrove environment.
Bacterial classification on the basis of a polyphasic approach was conducted on three poly(3 hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] accumulating bacterial strains that were isolated from samples collected from Malaysian environments; Kulim Lake, Sg. Pinang river and Sg. Manik paddy field. The Gram-negative, rod-shaped, motile, non-sporulating and non-fermenting bacteria were shown to belong to the genus Cupriavidus of the Betaproteobacteria on the basis of their 16S rRNA gene sequence analyses. The sequence similarity value with their near phylogenetic neighbour, Cupriavidus pauculus LMG3413T, was 98.5%. However, the DNA-DNA hybridization values (8-58%) and ribotyping analysis both enabled these strains to be differentiated from related Cupriavidus species with validly published names. The RiboPrint patterns of the three strains also revealed that the strains were genetically related even though they displayed a clonal diversity. The major cellular fatty acids detected in these strains included C15:0 ISO 2OH/C16:1 ω7c, hexadecanoic (16:0) and cis-11-octadecenoic (C18:1 ω7c). Their G+C contents ranged from 68.0 to 68.6 mol%, and their major isoprenoid quinone was Ubiquinone Q-8. Of these three strains, only strain USMAHM13 (= DSM 25816 = KCTC 32390) was discovered to exhibit yellow pigmentation that is characteristic of the carotenoid family. Their assembled genomes also showed that the three strains were not identical in terms of their genome sizes that were 7.82, 7.95 and 8.70 Mb for strains USMAHM13, USMAA1020 and USMAA2-4, respectively, which are slightly larger than that of Cupriavidus necator H16 (7.42 Mb). The average nucleotide identity (ANI) results indicated that the strains were genetically related and the genome pairs belong to the same species. On the basis of the results obtained in this study, the three strains are considered to represent a novel species for which the name Cupriavidus malaysiensis sp. nov. is proposed. The type strain of the species is USMAA1020T (= DSM 19416T = KCTC 32390T).
A novel, rod-shaped, Gram-stain-negative, halophilic and non-motile bacterium, designated CCB-MM1T, was isolated from a sample of estuarine sediment collected from Matang Mangrove Forest, Malaysia. The cells possessed a rod-coccus cell cycle in association with growth phase and formed aggregates. Strain CCB-MM1T was both catalase and oxidase positive, and able to degrade starch. Optimum growth occurred at 30 °C and pH 7.0 in the presence of 2-3 % (w/v) NaCl. The 16S rRNA gene sequence of strain CCB-MM1T showed 98.12, 97.46 and 97.33 % sequence similarity with Microbulbifer rhizosphaerae Cs16bT, Microbulbifer maritimus TF-17T and Microbulbifergwangyangensis GY2T respectively. Strain CCB-MM1T and M. rhizosphaerae Cs16bT formed a cluster in the phylogenetic tree. The major cellular fatty acids were iso-C17 : 1 ω9c and iso-C15 : 0, and the total polar lipid profile consisted of phosphatidylglycerol, phosphatidylethanolamine, phosphoaminolipid, two unidentified lipids, an unidentified glycolipid and an unidentified aminolipid. The major respiratory quinone was ubiquinone Q-8 and the genomic DNA G+C content of the strain was 58.9 mol%. On the basis of the phylogenetic, phenotypic and genotypic data presented here, strain CCB-MM1T represents a novel species of the genus Microbulbifer, for which the name Microbulbiferaggregans sp. nov. is proposed. The type strain is CCB-MM1T (=LMG 29920T=JCM 31875T).
Microbulbifer sp. CCB-MM1 is a halophile isolated from estuarine sediment of Matang Mangrove Forest, Malaysia. Based on 16S rRNA gene sequence analysis, strain CCB-MM1 is a potentially new species of genus Microbulbifer. Here we describe its features and present its complete genome sequence with annotation. The genome sequence is 3.86 Mb in size with GC content of 58.85%, harbouring 3313 protein coding genes and 92 RNA genes. A total of 71 genes associated with carbohydrate active enzymes were found using dbCAN. Ectoine biosynthetic genes, ectABC operon and ask_ect were detected using antiSMASH 3.0. Cell shape determination genes, mreBCD operon, rodA and rodZ were annotated, congruent with the rod-coccus cell cycle of the strain CCB-MM1. In addition, putative mreBCD operon regulatory gene, bolA was detected, which might be associated with the regulation of rod-coccus cell cycle observed from the strain.
In the original publication of the article, it was noted that Fig. 1 present an image of Cupriavidus malaysiensis strain USMAHM13 and not of strain USMAA1020, as indicated in the figure legend. The image in the original version is thus noted to serve as a record of strain USMAHM13 and the corrected version of Fig. 1 is reprinted below.
Poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] is a bacterial derived biopolymer widely known for its unique physical and mechanical properties to be used in biomedical application. In this study, antimicrobial agent silver sulfadiazine (SSD) coat/collagen peptide coat-P(3HB-co-4HB) (SCCC) and SSD blend/collagen peptide coat-P(3HB-co-4HB) scaffolds (SBCC) were fabricated using a green salt leaching technique combined with freeze-drying. This was then followed by the incorporation of collagen peptides at various concentrations (2.5-12.5 wt.%) to P(3HB-co-4HB) using collagen-coating. As a result, two types of P(3HB-co-4HB) scaffolds were fabricated, including SCCC and SBCC scaffolds. The increasing concentrations of collagen peptides from 2.5 wt.% to 12.5 wt.% exhibited a decline in their porosity. The wettability and hydrophilicity increased as the concentration of collagen peptides in the scaffolds increased. In terms of the cytotoxic results, MTS assay demonstrated the L929 fibroblast scaffolds adhered well to the fabricated scaffolds. The 10 wt.% collagen peptides coated SCCC and SBCC scaffolds displayed highest cell proliferation rate. The antimicrobial analysis of the fabricated scaffolds exhibited 100% inhibition towards various pathogenic microorganisms. However, the SCCC scaffold exhibited 100% inhibition between 12 and 24 h, but the SBCC scaffolds with SSD impregnated in the scaffold had controlled release of the antimicrobial agent. Thus, this study will elucidate the surface interface-cell interactions of the SSD-P(3HB-co-4HB)-collagen peptide scaffolds and controlled release of SSD, antimicrobial agent.
Depolymerase is an enzyme that plays an important role in the hydrolysis of polyhydroxyalkanoates [PHAs]. In the current study, Burkholderia cepacia DP1 was obtained from Penang, Malaysia in which the enzyme was purified using ion exchange and gel filtration (Superdex-75) column chromatography. The molecular mass of the enzyme was estimated to be 53.3 kDa using SDS-PAGE. The enzyme activity was increased to 36.8 folds with the recovery of 16.3% after purification. The enzyme activity was detected between pH 6.0-10 and at 35-55 °C with pH 6.0 and 45 °C facilitating the maximum activity. Depolymerase was inactivated by Tween-20, Tween-80, SDS and PMSF, but insensitive to metal ions (Mg2+, Ca2+, K+, Na2+, Fe3+) and organic solvents (methanol, ethanol, and acetone). The apparent Km values of the purified P(3HB) depolymerase enzyme for P(3HB) and P(3HB-co-14%3HV) were 0.7 mg/ml and 0.8 mg/ml, respectively. The Vmax values of the purified enzyme were 10 mg/min and 8.89 mg/min for P(3HB) and P(3HB-co-14%3HV), respectively. The current study discovered a new extracellular poly(3-hydroxybutyrate) [P(3HB)] depolymerase enzyme from Burkholderia cepacia DP1 isolated and purified to homogeneity from the culture supernatant. To the best of our knowledge, this is the first report demonstrating the purification and biochemical characterization of P(3HB) depolymerase enzyme from genus Burkholderia.
The global transplantation market size was valued at USD 8.4 billion in 2020 and is expected to grow at a compound annual growth rate of 11.5% over the forecast period. The increasing demand for tissue transplantation has inspired researchers to find alternative approaches for making artificial tissues and organs function. The unique physicochemical and biological properties of biopolymers and the attractive structural characteristics of aerogels such as extremely high porosity, ultra low-density, and high surface area make combining these materials of great interest in tissue scaffolding and regenerative medicine applications. Numerous biopolymer aerogel scaffolds have been used to regenerate skin, cartilage, bone, and even heart valves and blood vessels by growing desired cells together with the growth factor in tissue engineering scaffolds. This review focuses on the principle of tissue engineering and regenerative medicine and the role of biopolymer aerogel scaffolds in this field, going through the properties and the desirable characteristics of biopolymers and biopolymer tissue scaffolds in tissue engineering applications. The recent advances of using biopolymer aerogel scaffolds in the regeneration of skin, cartilage, bone, and heart valves are also discussed in the present review. Finally, we highlight the main challenges of biopolymer-based scaffolds and the prospects of using these materials in regenerative medicine.