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  1. Raveena, K., Arularasu, M., Ganesan, S.S. K., Amelia, T.S.M., Ong, M.C., Bhubalan, K., et al.
    MyJurnal
    Mangrove located near urban area is exposed to various industrial discharge including heavy metals. Mangrove soil is capable of accumulating and storing these heavy metals. Heavy metals are toxic and non-biodegradable, so their accumulations affect water quality, while bioaccumulation and bio-assimilation of heavy metals in mangrove organisms negatively impact the food chain. Bacteria-derived biosurfactants are compounds capable of removing heavy metals from soil and sediment. Furthermore, environmentally friendly properties, such as biodegradability and low toxicity, exhibited by biosurfactants make them a suitable replacement for chemical surfactants for remediation efforts. This study was conducted to investigate the lead- (Pb) and zinc- (Zn) removing capability of rhamnolipid (RL), a type of biosurfactant produced by marine bacterium, Pseudomonas aeruginosa UMTKB-5. Rhamnolipid solutions of three different concentrations (25 mg/L, 50 mg/L and 75 mg/L) were added to mangrove soil and incubated for 7 days. The removal of Pb from soils was up to 18.3% using 25 mg/L RL solution, while 50 mg/L RL solution removed 48.3%, and 75 mg/L RL solution removed 75.9% Pb over time. Meanwhile, zinc removal of 25 mg/L RL solution was up to 24.9%, while 50 mg/L removed 16.5%, and 75 mg/L RL removed 30.5% of Zn. The results showed that RL from P. aeruginosa UMTKB-5 could be a potential biomaterial to be used to remediate heavy metals in sediment.
  2. Vigneswari S, Lee TS, Bhubalan K, Amirul AA
    Enzyme Res, 2015;2015:212159.
    PMID: 26664741 DOI: 10.1155/2015/212159
    Bacteria capable of degrading polyhydroxyalkanoates (PHA) by secreting extracellular depolymerase enzymes were isolated from water and soil samples collected from various environments in Malaysia. A total of 8 potential degraders exhibited clear zones on poly(3-hydroxybutyrate) [P(3HB)] based agar, indicating the presence of extracellular PHA depolymerase. Among the isolates, DP5 exhibited the largest clearing zone with a degradation index of 6.0. The highest degradation activity of P(3HB) was also observed with depolymerase enzyme of DP5 in mineral salt medium containing P(3HB). Based on biochemical characterization and 16S rRNA cloning and sequencing, isolate DP5 was found to belong to the genus Acidovorax and subsequently named as Acidovorax sp. DP5. The highest extracellular depolymerase enzyme activity was achieved when 0.25% (w/v) of P(3HB) and 1 g/L of urea were used as carbon and nitrogen source, respectively, in the culture media. The most suitable assay condition of the depolymerase enzyme in response to pH and temperature was tested. The depolymerase produced by strain Acidovorax sp. DP5 showed high percentage of degradation with P(3HB) films in an alkaline condition with pH 9 and at a temperature of 40°C.
  3. Amelia TSM, Amirul AA, Bhubalan K
    Data Brief, 2018 Feb;16:75-80.
    PMID: 29188224 DOI: 10.1016/j.dib.2017.11.011
    We report data associated with the identification of three polyhydroxyalkanoate synthase genes (phaC) isolated from the marine bacteria metagenome of Aaptos aaptos marine sponge in the waters of Bidong Island, Terengganu, Malaysia. Our data describe the extraction of bacterial metagenome from sponge tissue, measurement of purity and concentration of extracted metagenome, polymerase chain reaction (PCR)-mediated amplification using degenerate primers targeting Class I and II phaC genes, sequencing at First BASE Laboratories Sdn Bhd, and phylogenetic analysis of identified and known phaC genes. The partial nucleotide sequences were aligned, refined, compared with the Basic Local Alignment Search Tool (BLAST) databases, and released online in GenBank. The data include the identified partial putative phaC and their GenBank accession numbers, which are Rhodocista sp. phaC (MF457754), Pseudomonas sp. phaC (MF437016), and an uncultured bacterium AR5-9d_16 phaC (MF457753).
  4. Amelia TSM, Lau NS, Amirul AA, Bhubalan K
    Data Brief, 2020 Aug;31:105971.
    PMID: 32685631 DOI: 10.1016/j.dib.2020.105971
    Marine sponges are acknowledged as a bacterial hotspot and resource of novel natural products or genetic material with industrial or commercial potential. However, sponge-associated bacteria are difficult to be cultivated and the production of their desirable metabolites is inadequate in terms of rate and quantity, yet bioinformatics and metagenomics tools are steadily progressing. Bacterial diversity profiles of high-microbial-abundance wild tropical marine sponges Aaptos aaptos and Xestospongia muta were obtained by sample collection at Pulau Bidong and Pulau Redang islands, 16S rRNA amplicon sequencing on Illumina HiSeq2500 platform (250 bp paired-end) and metagenomics analysis using Ribosomal Database Project (RDP) classifier. Raw sequencing data in fastq format and relative abundance histograms of the dominant 10 species are available in the public repository Discover Mendeley Data (http://dx.doi.org/10.17632/zrcks5s8xp). Filtered sequencing data of operational taxonomic unit (OTU) with chimera removed is available in NCBI accession numbers from MT464469 to MT465036.
  5. Amelia TSM, Amirul AA, Saidin J, Bhubalan K
    Trop Life Sci Res, 2018 Jul;29(2):187-199.
    PMID: 30112149 MyJurnal DOI: 10.21315/tlsr2018.29.2.13
    Marine sponges are acknowledged as bacterial hotspots in the oceanic biome. Aquatic bacteria are being investigated comprehensively for bioactive complexes and secondary metabolites. Cultivable bacteria associated with different species of sea sponges in South China Sea waters adjacent to Bidong Island, Terengganu were identified. Molecular identification was accomplished using 16S rRNA gene cloning and sequencing. Fourteen bacterial species were identified and their phylogenetic relationships were analysed by constructing a neighbour-joining tree with Molecular Evolutionary Genetics Analysis 6. The identified species encompassed four bacterial classes that were Firmicutes, Actinobacteria, Alphaproteobacteria and Gammaproteobacteria known to have been associated with sponges. The potential biotechnological applications of the identified bacteria were compared and reviewed based on relevant past studies. The biotechnological functions of the 14 cultivable isolates have been previously reported, hence reinforcing that bacteria associated with sponges are an abundant resource of scientifically essential compounds. Resilience of psychrotolerant bacteria, Psychrobacter celer, in warm tropical waters holds notable prospects for future research.
  6. Sudesh K, Bhubalan K, Chuah JA, Kek YK, Kamilah H, Sridewi N, et al.
    Appl Microbiol Biotechnol, 2011 Mar;89(5):1373-86.
    PMID: 21279347 DOI: 10.1007/s00253-011-3098-5
    Polyhydroxyalkanoate (PHA) is a potential substitute for some petrochemical-based plastics. This biodegradable plastic is derived from microbial fermentation using various carbon substrates. Since carbon source has been identified as one of the major cost-absorbing factors in PHA production, cheap and renewable substrates are currently being investigated as substitutes for existing sugar-based feedstock. Plant oils have been found to result in high-yield PHA production. Malaysia, being the world's second largest producer of palm oil, is able to ensure continuous supply of palm oil products for sustainable PHA production. The biosynthesis and characterization of various types of PHA using palm oil products have been described in detail in this review. Besides, by-products and waste stream from palm oil industry have also demonstrated promising results as carbon sources for PHA biosynthesis. Some new applications in cosmetic and wastewater treatment show the diversity of PHA usage. With proper management practices and efficient milling processes, it may be possible to supply enough palm oil-based raw materials for human consumption and other biotechnological applications such as production of PHA in a sustainable manner.
  7. Vigneswari S, Amelia TSM, Hazwan MH, Mouriya GK, Bhubalan K, Amirul AA, et al.
    Antibiotics (Basel), 2021 Feb 24;10(3).
    PMID: 33668352 DOI: 10.3390/antibiotics10030229
    Nanobiotechnology has undoubtedly influenced major breakthroughs in medical sciences. Application of nanosized materials has made it possible for researchers to investigate a broad spectrum of treatments for diseases with minimally invasive procedures. Silver nanoparticles (AgNPs) have been a subject of investigation for numerous applications in agriculture, water treatment, biosensors, textiles, and the food industry as well as in the medical field, mainly due to their antimicrobial properties and nanoparticle nature. In general, AgNPs are known for their superior physical, chemical, and biological properties. The properties of AgNPs differ based on their methods of synthesis and to date, the biological method has been preferred because it is rapid, nontoxic, and can produce well-defined size and morphology under optimized conditions. Nevertheless, the common issue concerning biological or biobased production is its sustainability. Researchers have employed various strategies in addressing this shortcoming, such as recently testing agricultural biowastes such as fruit peels for the synthesis of AgNPs. The use of biowastes is definitely cost-effective and eco-friendly; moreover, it has been reported that the reduction process is simple and rapid with reasonably high yield. This review aims to address the developments in using fruit- and vegetable-based biowastes for biologically producing AgNPs to be applied as antimicrobial coatings in biomedical applications.
  8. Govindasamy S, Syafiq IM, Amirul AA, Amin RM, Bhubalan K
    Data Brief, 2019 Apr;23:103675.
    PMID: 30788397 DOI: 10.1016/j.dib.2019.01.023
    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.
  9. Kee SH, Chiongson JBV, Saludes JP, Vigneswari S, Ramakrishna S, Bhubalan K
    Environ Pollut, 2021 Feb 15;271:116311.
    PMID: 33383425 DOI: 10.1016/j.envpol.2020.116311
    Global increase in demand for food supply has resulted in surplus generation of wastes. What was once considered wastes, has now become a resource. Studies were carried out on the conversion of biowastes into wealth using methods such as extraction, incineration and microbial intervention. Agro-industry biowastes are promising sources of carbon for microbial fermentation to be transformed into value-added products. In the era of circular economy, the goal is to establish an economic system which aims to eliminate waste and ensure continual use of resources in a close-loop cycle. Biowaste collection is technically and economically practicable, hence it serves as a renewable carbon feedstock. Biowastes are commonly biotransformed into value-added materials such as bioethanol, bioplastics, biofuels, biohydrogen, biobutanol and biogas. This review reveals the recent developments on microbial transformation of biowastes into biotechnologically important products. This approach addresses measures taken globally to valorize waste to achieve low carbon economy. The sustainable use of these renewable resources is a positive approach towards waste management and promoting circular economy.
  10. Syafiq IM, Huong KH, Shantini K, Vigneswari S, Aziz NA, Amirul AA, et al.
    Enzyme Microb Technol, 2017 Mar;98:1-8.
    PMID: 28110659 DOI: 10.1016/j.enzmictec.2016.11.011
    Poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] copolymer is noted for its high biocompatibility, which makes it an excellent candidate for biopharmaceutical applications. The wild-type Cupriavidus sp. USMAA1020 strain is able to synthesize P(3HB-co-4HB) copolymers with different 4HB monomer compositions (up to 70mol%) in shaken flask cultures. Combinations of 4HB carbon precursors consisting of 1,6-hexanediol and γ-butyrolactone were applied for the production of P(3HB-co-4HB) with different 4HB molar fraction. A sharp increase in 4HB monomer composition was attained by introducing additional copies of PHA synthase gene (phaC), responsible for P(3HB-co-4HB) polymerization. The phaC of Cupriavidus sp. USMAA1020 and Cupriavidus sp. USMAA2-4 were cloned and heterologously introduced into host, wild-type Cupriavidus sp. USMAA1020. The gene dosage treatment resulted in the accumulation of 93mol% 4HB by the transformant strains when grown in similar conditions as the wild-type USMAA1020. The PHA synthase activities for both transformants were almost two-fold higher than the wild-type. The ability of the transformants to produce copolymers with high 4HB monomer composition was also tested in large scale production system using 5L and 30L bioreactors with a constant oxygen mass transfer rate. The 4HB monomer composition could be maintained at a range of 83-89mol%. The mechanical and thermal properties of copolymers improved with increasing 4HB monomer composition. The copolymers produced could be tailored for specific biopharmaceutical applications based on their properties.
  11. Mohamed S, May Amelia TS, Abdullah Amirul AA, Abdul Wahid ME, Bhubalan K
    Biologicals, 2021 Jun;71:51-54.
    PMID: 33858743 DOI: 10.1016/j.biologicals.2021.03.002
    A natural biodegradable polymer, polyhydroxyalkanoate (PHA), was adjuvanted with a vaccine seed to observe the biomaterial's ability in enhancing an immune response in rats. The adjuvant potential of PHA was tested using the whole-killed Pasteurella multocida B:2 (PMB2) vaccine in Sprague Dawley (SD) rats to detect changes in serum immunoglobulin G (IgG) and immunoglobulin M (IgM) responses. A common PHA, poly(3-hydroxybutyrate) [P(3HB)], from Bacillus megaterium UMTKB-1 was constructed into microparticles using the solvent evaporation method. Twelve SD rats were divided into four treatment groups: 1) non-treatment as negative control, 2) P(3HB) adjuvant, 3) PMB2 vaccine, and 4) adjuvanted-P(3HB)/PMB2 vaccine groups, which were intramuscularly vaccinated twice. Immunoglobulins IgG and IgM levels were used as markers of the immune response induced by the adjuvanted-P(3HB)/PMB2 vaccine and analysed over an eight-week study period. The group vaccinated specifically with adjuvanted-P(3HB)/PMB2 vaccine had higher concentrations of immunoglobulins compared to other treatment groups, hence demonstrating the potential of the adjuvant to enhance immune response. Findings showed a need to delay the delivery of the second booster dose to determine the appropriate regime for the adjuvanted-P(3HB)/PMB2 vaccine.
  12. Bhubalan K, Chuah JA, Shozui F, Brigham CJ, Taguchi S, Sinskey AJ, et al.
    Appl Environ Microbiol, 2011 May;77(9):2926-33.
    PMID: 21398494 DOI: 10.1128/AEM.01997-10
    The synthesis of bacterial polyhydroxyalkanoates (PHA) is very much dependent on the expression and activity of a key enzyme, PHA synthase (PhaC). Many efforts are being pursued to enhance the activity and broaden the substrate specificity of PhaC. Here, we report the identification of a highly active wild-type PhaC belonging to the recently isolated Chromobacterium sp. USM2 (PhaC(Cs)). PhaC(Cs) showed the ability to utilize 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV), and 3-hydroxyhexanoate (3HHx) monomers in PHA biosynthesis. An in vitro assay of recombinant PhaC(Cs) expressed in Escherichia coli showed that its polymerization of 3-hydroxybutyryl-coenzyme A activity was nearly 8-fold higher (2,462 ± 80 U/g) than that of the synthase from the model strain C. necator (307 ± 24 U/g). Specific activity using a Strep2-tagged, purified PhaC(Cs) was 238 ± 98 U/mg, almost 5-fold higher than findings of previous studies using purified PhaC from C. necator. Efficient poly(3-hydroxybutyrate) [P(3HB)] accumulation in Escherichia coli expressing PhaC(Cs) of up to 76 ± 2 weight percent was observed within 24 h of cultivation. To date, this is the highest activity reported for a purified PHA synthase. PhaC(Cs) is a naturally occurring, highly active PHA synthase with superior polymerizing ability.
  13. Vigneswari S, Noor MSM, Amelia TSM, Balakrishnan K, Adnan A, Bhubalan K, et al.
    Life (Basel), 2021 Aug 10;11(8).
    PMID: 34440551 DOI: 10.3390/life11080807
    Polyhydroxyalkanoates (PHA) are biodegradable polymers that are considered able to replace synthetic plastic because their biochemical characteristics are in some cases the same as other biodegradable polymers. However, due to the disadvantages of costly and non-renewable carbon sources, the production of PHA has been lower in the industrial sector against conventional plastics. At the same time, first-generation sugar-based cultivated feedstocks as substrates for PHA production threatens food security and considerably require other resources such as land and energy. Therefore, attempts have been made in pursuit of suitable sustainable and affordable sources of carbon to reduce production costs. Thus, in this review, we highlight utilising waste lignocellulosic feedstocks (LF) as a renewable and inexpensive carbon source to produce PHA. These waste feedstocks, second-generation plant lignocellulosic biomass, such as maize stoves, dedicated energy crops, rice straws, wood chips, are commonly available renewable biomass sources with a steady supply of about 150 billion tonnes per year of global yield. The generation of PHA from lignocellulose is still in its infancy, hence more screening of lignocellulosic materials and improvements in downstream processing and substrate pre-treatment are needed in the future to further advance the biopolymer sector.
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