A novel wastewater treatment system consisting of an up-flow anaerobic sludge blanket (UASB) reactor and a down-flow hanging sponge (DHS) reactor with sulfur-redox reaction was developed for treatment of municipal sewage under low-temperature conditions. In the UASB reactor, a novel phenomenon of anaerobic sulfur oxidation occurred in the absence of oxygen, nitrite and nitrate as electron acceptors. The microorganisms involved in anaerobic sulfur oxidation have not been elucidated. Therefore, in this study, we studied the microbial communities existing in the UASB reactor that probably enhanced anaerobic sulfur oxidation. Sludge samples collected from the UASB reactor before and after sulfur oxidation were used for cloning and terminal restriction fragment length polymorphism (T-RFLP) analysis of the 16S rRNA genes of the bacterial and archaeal domains. The microbial community structures of bacteria and archaea indicated that the genus Smithella and uncultured bacteria within the phylum Caldiserica were the dominant bacteria groups. Methanosaeta spp. was the dominant group of the domain archaea. The T-RFLP analysis, which was consistent with the cloning results, also yielded characteristic fingerprints for bacterial communities, whereas the archaeal community structure yielded stable microbial community. From these results, it can be presumed that these major bacteria groups, genus Smithella and uncultured bacteria within the phylum Caldiserica, probably play an important role in sulfur oxidation in UASB reactors.
An obligate anaerobic bacterium Clostridium difficile has a unique metabolic pathway to convert leucine to 4-methylvalerate, in which 4-methyl-2-pentenoyl-CoA (4M2PE-CoA) is an intermediate of this pathway. 4M2PE-CoA is also able to be converted to 3-hydroxy-4-methylvalerate (3H4MV), a branched side chain monomer unit, for synthesis of polyhydroxyalkanoate (PHA) copolymer. In this study, to synthesize 3H4MV-containing PHA copolymer from leucine, the leucine metabolism-related enzymes (LdhA and HadAIBC) derived from C. difficile and PHA biosynthesis enzymes (PhaPCJAc and PhaABRe) derived from Aeromonas caviae and Ralstonia eutropha were co-expressed in the codon usage-improved Escherichia coli. Under microaerobic culture conditions, this E. coli was able to synthesize P(3HB-co-12.2 mol% 3H4MV) from glucose with the supplementation of 1 g/L leucine. This strain also produced P(3HB-co-12.6 mol% 3H4MV) using the culture supernatant of leucine overproducer E. coli strain NS1391 as the medium for PHA production, achieving 3H4MV copolymer synthesis only from glucose. Furthermore, we tested the feasibility of the 3H4MV copolymer synthesis in E. coli strain NS1391 from glucose. The recombinant E. coli NS1391 was able to synthesize P(3HB-co-3.0 mol% 3H4MV) from glucose without any leucine supplementation. This study demonstrates the potential of the new metabolic pathway for 3H4MV synthesis using leucine metabolism-related enzymes from C. difficile.
A gel imaging method was employed to quantitate the GFP that had been subjected to denaturation and degradation treatments. This method is able to differentiate the nativity of GFP by relating the observed changes in the position of fluorescent bands which is unable to be detected using the spectrofluorometric method.
Serum deprivation inhibits cell growth and initiates apoptosis cell death in mammalian cell cultures. Since apoptosis is a genetically controlled cell death pathway, over-expression of anti-apoptotic proteins may provide a way to delay apoptosis. This study investigated the ability of the X-linked inhibitor of apoptosis protein (XIAP) to inhibit apoptosis induced by serum deprivation. Study includes evaluation of the ability of XIAP to prolong culture period and its effect on cell proliferation in serum-deprived media. The full length human XIAP was introduced into CHO-K1 cell lines and the effects of XIAP over-expression on the inhibition of apoptosis induced by serum-deprived conditions were examined. In batch cultures, cells over-expressing XIAP showed decreased levels of apoptosis and a higher number of viable cell under serum-deprived conditions compared to the control cell lines. The viability of control cells dropped to 40% after 2days of serum deprivation, the XIAP expressing cells still maintained at a viability higher than 90%. Further investigation revealed that the caspase-3 activity of the CHO-K1 cell line was inhibited as a result of XIAP expression.
Amauroderma rugosum is a wild mushroom species widely distributed in tropics and is classified under the class of Basidiomycetes. Basidiomycetes are well-known for their abilities of producing lignocellulolytic enzymes such as lignin peroxidase (LiP), laccase (Lac) and manganese peroxidase (MnP). Different factors such as nutrient sources, incubation period and agitation affect the production of lignocellulolytic enzymes. The A. rugosum produced LiP in the medium supplemented with potato dextrose broth (PDB), 0.5% yeast and 1.0% saw dust at 26.70±3.31 U/mL. However, the LiP activity was increased to 106.32±5.32 U/mL when supplemented with 150 μm of copper (CuSO4). The aqueous two-phase system (ATPS) is a simple, rapid and low cost method for primary extraction and recovery of LiP. A total of 25 systems made from five different molecular weights of polyethylene glycol (PEG)/dipotassium hydrogen phosphate (K2HPO4) were tested. PEG 600 produced the highest top phase purification factor (PFT) of 1.33±0.62 with yield of 72.18±8.50%. The optimization of the ATPS parameters, such as volume ratio VR, pH and crude enzyme loading are the factors controlling the phase partition. Our results showed that significant improvement (PFT of 6.26±2.87 with yield of 87.31±3.14%) of LiP recovery can be achieved by optimized the parameters.
The C-terminal domain of Nipah virus (NiV) nucleocapsid protein (NP₄₀₁₋₅₃₂) was inserted at the N-terminus and the immunodominant loop of hepatitis B core antigen (HBc). The stability of NP₄₀₁₋₅₃₂ increased tremendously when displayed on the HBc particles. These particles reacted specifically with the swine anti-NiV and the human anti-HBc antisera.
PHA synthase (PhaC) is the key enzyme in the production of biodegradable plastics known as polyhydroxyalkanoate (PHA). Nevertheless, most of these enzymes are isolated from cultivable bacteria using traditional isolation method. Most of the microorganisms found in nature could not be successfully cultivated due to the lack of knowledge on their growth conditions. In this study, a culture-independent approach was applied. The presence of phaC genes in limestone soil was screened using primers targeting the class I and II PHA synthases. Based on the partial gene sequences, a total of 19 gene clusters have been identified and 7 clones were selected for full length amplification through genome walking. The complete phaC gene sequence of one of the clones (SC8) was obtained and it revealed 81% nucleotide identity to the PHA synthase gene of Chromobacterium violaceum ATCC 12472. This gene obtained from uncultured bacterium was successfully cloned and expressed in a Cupriavidus necator PHB(-)4 PHA-negative mutant resulting in the accumulation of significant amount of PHA. The PHA synthase activity of this transformant was 64 ± 12 U/g proteins. This paper presents a pioneering study on the discovery of phaC in a limestone area using metagenomic approach. Through this study, a new functional phaC was discovered from uncultured bacterium. Phylogenetic classification for all the phaCs isolated from this study has revealed that limestone hill harbors a great diversity of PhaCs with activities that have not yet been investigated.
Aquitalea sp. USM4 (JCM 19919) was isolated from a freshwater sample at Lata Iskandar Waterfall in Perak, Malaysia. It is a rod-shaped, gram-negative bacterium with high sequence identity (99%) to Aquitalea magnusonii based on 16S rRNA gene analysis. Aquitalea sp. USM4 also possessed a PHA synthase gene (phaC), which had amino acid sequence identity of 77-78% to the PHA synthase of Chromobacterium violaceum ATCC12472 and Pseudogulbenkiania sp. NH8B. PHA biosynthesis results showed that wild-type Aquitalea sp. USM4 was able to accumulate up to 1.5 g/L of poly(3-hydroxybutyrate), [P(3HB)]. The heterologous expression of the PHA synthase gene of Aquitalea sp. USM4 (phaCAq) in Cupriavidus necator PHB(-)4 had resulted in PHA accumulation up to 3.2 g/L of P(3HB). It was further confirmed by (1)H nuclear magnetic resonance (NMR) analysis that Aquitalea sp. USM4 and C. necator PHB(-)4 transformant were able to produce PHA containing 3-hydroxyvalerate (3HV), 4-hydroxybutyrate (4HB) and 3-hydroxy-4-methylvalerate (3H4MV) monomers from suitable precursor substrates. Interestingly, relatively high PHA synthase activity of 863 U/g and 1402 U/g were determined in wild-type Aquitalea sp. USM4 and C. necator PHB(-)4 transformant respectively. This is the first report on the member of genus Aquitalea as a new PHA producer as well as in vitro and in vivo characterization of a novel PHA synthase from Aquitalea sp. USM4.
Phosphoric acid impregnated activated carbon from date pits (DPAC) was prepared through single step activation. Prepared DPAC was studied for its structural, elemental, chemical, surface and crystal nature. Adsorption ability of the DPAC was assessed through divalent lead ions separation studies. Effect of adsorbent dosage, contact time, pH, operating temperature and initial feed concentration on lead removal by DPAC was studied. Maximum Pb(II) adsorption capacity of 101.35 mg/g was attained for a contact time of 30 min and pH of 6 at 30°C. Increase in initial feed concentration enhanced the adsorption ability of DPAC and the rise in adsorbent dosage resulted in improved Pb(II) removal efficiency. Thermodynamic studies revealed that the lead adsorption on DPAC was exothermic and instantaneous in nature. Kinetic and equilibrium studies confirmed the suitability of pseudo-second order and Langmuir isotherm for divalent lead ions binding on DPAC. Reusability studies showed that HCl was the effective regeneration medium and the DPAC could be reused for a maximum of 4 times with slight reduction in Pb(II) removal efficiency (<10%). Results indicated the promising use of date pits biomass as a low cost and efficient starting material to prepare activated carbon for divalent lead ions removal.
As one of Lianyungang's most famous specialties, Acanthogobius hasta is delicious and nutritious fish, but is extremely susceptible to spoilage during transportation and storage. In this study, Lactobacillus plantarum MMB-07 was isolated from traditional fermented sour fish to reduce losses and improve the utilization and food value of A. hasta. L. plantarum MMB-07 had good ability of acid production and acid resistance. Moreover, it could also inhibit common pathogens in food or aquatic products to ensure the safety of fermented products. MMB-07 was used to ferment A. hasta and obtain fermented Suanyu rich in nutrition value and good flavor. The volatile base nitrogen was 18.44 mg/100 g and the fermented fish meat maintained second-grade freshness. Thiobarbituric acid assay was 0.90 mg/kg and fat in fish meat was oxidized to a low degree. The studies indicated that MMB-07 has a high application prospect in low salt fermented fish.
Polyhydroxyalkanoates (PHAs), a class of renewable and biodegradable green polymers, have gained attraction as a potential substitute for the conventional plastics due to the increasing concern towards environmental pollution as well as the rapidly depleting petroleum reserve. Nevertheless, the high cost of downstream processing of PHA has been a bottleneck for the wide adoption of PHAs. Among the options of PHAs recovery techniques, aqueous two-phase extraction (ATPE) outshines the others by having the advantages of providing a mild environment for bioseparation, being green and non-toxic, the capability to handle a large operating volume and easily scaled-up. Utilizing unique properties of thermo-responsive polymer which has decreasing solubility in its aqueous solution as the temperature rises, cloud point extraction (CPE) is an ATPE technique that allows its phase-forming component to be recycled and reused. A thorough literature review has shown that this is the first time isolation and recovery of PHAs from Cupriavidus necator H16 via CPE was reported. The optimum condition for PHAs extraction (recovery yield of 94.8% and purification factor of 1.42 fold) was achieved under the conditions of 20 wt/wt % ethylene oxide-propylene oxide (EOPO) with molecular weight of 3900 g/mol and 10 mM of sodium chloride addition at thermoseparating temperature of 60°C with crude feedstock limit of 37.5 wt/wt %. Recycling and reutilization of EOPO 3900 can be done at least twice with satisfying yield and PF. CPE has been demonstrated as an effective technique for the extraction of PHAs from microbial crude culture.
In this study, endoglucanase was produced from oil palm empty fruit bunch (OPEFB) by a locally isolated aerobic bacterium, Bacillus pumilus EB3. The effects of the fermentation parameters such as initial pH, temperature, and nitrogen source on the endoglucanase production were studied using carboxymethyl cellulose (CMC) as the carbon source. Endoglucanase from B. pumilus EB3 was maximally secreted at 37 degrees C, initial pH 7.0 with 10 g/l of CMC as carbon source, and 2 g/l of yeast extract as organic nitrogen source. The activity recorded during the fermentation was 0.076 U/ml. The productivity of the enzyme increased twofold when 2 g/l of yeast extract was used as the organic nitrogen supplement as compared to the non-supplemented medium. An interesting finding from this study is that pretreated OPEFB medium showed comparable results to CMC medium in terms of enzyme production with an activity of 0.063 U/ml. As OPEFB is an abundant solid waste at palm oil mills, it has the potential of acting as a substrate in cellulase production.
Palm oil is a representative and important biomass, not only as the most edible vegetable oil consumed worldwide, but also as a material for chemicals and biofuels. Despite the potential sustainability of the palm oil industry, it has conventionally emitted excess greenhouse gases, waste materials, and wastewater, brought land use change, thus affecting the natural environment. Therefore, the successful development of a sustainable palm oil industry is a touchstone for promoting the bioeconomy. Here, we first review the concept of the bioeconomy and the positive and negative aspects of the palm oil industry. Then, we consider solutions for introducing a green economy into the palm oil industry, such that it may coexist with biodiversity and environmental conservation toward the Sustainable Development Goals.
Previously, a unique co-compost produced by composting empty fruit bunch with anaerobic sludge from palm oil mill effluent, which contributed to establishing a zero-emission industry in Malaysia. Little was known about the bacterial functions during the composting process and fertilization capacity of this co-compost. We isolated 100 strains from the co-compost on 7 types of enumeration media and screened 25 strains using in vitro tests for 12 traits, grouping them according to three functions: plant growth promoting (fixation of nitrogen; solubilization of phosphorus, potassium, and silicate; production of 3-indoleacetic acid, ammonia, and siderophore), biocontrolling (production of chitinase and anti-Ganoderma activity), and composting (degradation of lignin, xylan, and cellulose). Using 16S rRNA gene sequence analysis, 25 strains with strong or multi-functional traits were found belong to the genera Bacillus, Paenibacillus, Citrobacter, Enterobacter, and Kosakonia. Furthermore, several strains of Citrobacter sedlakii exhibited a plant growth-stimulation in vivo komatsuna plant cultivation test. In addition, we isolated several multifunctional strains; Bacillus tequilensis CE4 (biocontrolling and composting), Enterobacter cloacae subsp. dissolvens B3 (plant growth promoting and biocontrolling), and C. sedlakii CESi7 (plant growth promoting and composting). Some bacteria in the co-compost play significant roles during the composting process and plant cultivation after fertilization, and some multifunctional strains have potential for use in accelerating the biodegradation of lignocellulosic biomass, protecting against Ganoderma boninense infection, and increasing the yield of palm oil.
Alginate, a natural polysaccharide, was explored in this study as an oral delivery vehicle of a mammalian expression vector into the murine intestinal mucosa. Alginate microspheres were produced through water-in-oil (W/O) emulsification method. Average diameter sizes of microspheres were 46.88 μm±3.07 μm with significant size reduction upon utilization of 1.0% Span80. Plasmid DNA (pDNA) carrying green fluorescent protein reporter gene (GFP), pVAX-GFP, was encapsulated within microspheres at efficiencies of 72.9 to 74.4%, carrying maximum load of 6 μg pDNA. Alginate microspheres demonstrated shrinkage in pH 1.2 and swelling in pH 9.0 with pDNA release about twice the amount released in acidic environment. Oral delivery of pVAX-GFP loaded-microspheres, at 50 μg, 100 μg and 150 μg dose, was performed on BALB/c mice. Tissue biodistribution, investigated through flow cytometric analysis, demonstrated GFP positive intestinal cells (<1.0%) with 1.3-fold higher levels for the 100 μg dose; therefore suggesting feasibility of the approach for oral gene delivery and vaccination.
Fermented foods are gaining popularity due to health-promoting properties with high levels of nutrients, phytochemicals, bioactive compounds, and probiotic microorganisms. Due to its unique fermentation process, Lactococcus lactis plays a key role in the food business, notably in the manufacturing of dairy products. The superior biological activities of L. lactis in these functional foods include anti-inflammatory and immunomodulatory capabilities. L. lactis boosted growth performance, controlled amino acid profiles, intestinal immunology, and microbiota. Besides that, the administration of L. lactis increased the rate of infection clearance. Innate and acquired immune responses would be upregulated in both local and systemic compartments, resulting in these consequences. L. lactis is often employed in the food sector and is currently being exploited as a delivery vehicle for biological research. These bacteria are being eyed as potential candidates for biotechnological applications. With this in mind, we reviewed the immunomodulatory effects of different L. lactis strains.
The present study aimed to optimize the conditions for the production of adventitious roots from Eurycoma longifolia Jack, an important medicinal woody plant, in bioreactor culture. The effects of the type and concentration of auxin on root growth were studied, as well as the effects of the NH4(+):NO3(-) ratio on adventitious root growth and the production of phenolics and flavonoids. Approximately 5 g L(-1) fresh weight of adventitious roots was inoculated into a 3 L balloon-type bubble bioreactor, which contained 2 L 3/4 MS medium supplemented with 30 g L(-1) sucrose and cultures were maintained in the dark for 7 weeks at 24 ± 1°C. Higher concentrations of IBA (7.0 and 9.0 mg L(-1)) and NAA (5.0 mg L(-1)) enhanced the biomass and accumulation of total phenolics and flavonoids. The adventitious roots were thin, numerous, and elongated in 3/4 MS medium supplemented with 5.0 and 7.0 mg L(-1) IBA, whereas the lateral roots were shorter and thicker with 5.0 mg L(-1) NAA compared with IBA treatment. The optimum biomasses of 50.22 g L(-1) fresh weight and 4.60 g L(-1) dry weight were obtained with an NH4(+):NO3(-) ratio of 15:30. High phenolic and flavonoid productions (38.59 and 11.27 mg L(-1) medium, respectively) were also obtained with a ratio of 15:30. Analysis of the 2,2-diphenyl-1-picrylhydrazyl (DPPH)-scavenging activity indicated higher antioxidant activity with an NH4(+):NO3(-) ratio of 30:15. These results suggest that balloon-type bubble bioreactor cultures are suitable for the large-scale commercial production of E. longifolia adventitious roots which contain high yield of bioactive compounds.
In this present study, alcohol/salt liquid biphasic system was used to extract phlorotannin from brown macroalgae. Liquid biphasic system is a new green technology that integrated with various processes into one-step, by concentrating, separating and purifying the bioproduct in a unit operation. The solvent used is non-toxic and there is potential for solvent recovery which is beneficial to the environment. Phlorotannin is a bioactive compound that has gained much attention due to its health beneficial effect. Therefore, the isolation of phlorotannin is lucrative as it contains various biological activities that are capable to be utilised into food and pharmaceutical application. By using 2-propanol/ammonium sulphate system, the highest recovery of phlorotannin was 76.1% and 91.67% with purification factor of 2.49 and 1.59 from Padina australis and Sargassum binderi, respectively. A recycling study was performed and the salt phase of system was recycled where maximum salt recovery of 41.04% and 72.39% could be obtained from systems containing P. australis and S. binderi, respectively. Similar recovery of phlorotannin was observed after performing two cycles of the system, this concludes that the system has good recyclability and eco-friendly.
The effects of locally produced chitosan (CPSRT-NC-bicarbonate) in the intervention of keloid pathogenesis were investigated in vitro. A human keratinocyte-fibroblast co-culture model was established to investigate the protein levels of human collagen type-I, III and V in a western blotting analysis, the secreted transforming growth factor-β1 (TGF-β1) in an enzyme-linked immunosorbent assay (ELISA) and the mRNA levels of TGF-β1's intracellular signaling molecules (SMAD2, 3, 4 and 7) in a real-time PCR analysis. Keratinocyte-fibroblast co-cultures were maintained in DKSFM:DMEM:F12 (2:2:1) medium. Collagen type-I was found to be the dominant form in primary normal human dermal fibroblast (pNHDF) co-cultures, whereas collagen type-III was more abundant in primary keloid-derived human dermal fibroblast (pKHDF) co-cultures. Collagen type-V was present as a minor component in the skin. TGF-β1, SMAD2 and SMAD4 were expressed more in the pKHDF than the pNHDF co-cultures. Co-cultures with normal keratinocytes suppressed collagen type-III, SMAD2, SMAD4 and TGF-β1 expressions and CPSRT-NC-bicarbonate enhanced this effect. In conclusion, the CPSRT-NC-bicarbonate in association with normal-derived keratinocytes demonstrated an ability to reduce TGF-β1, SMAD2 and SMAD4 expressions in keloid-derived fibroblast cultures, which may be useful in keloid intervention.
Metabolic engineering is a research field that focuses on the design of models for metabolism, and uses computational procedures to suggest genetic manipulation. It aims to improve the yield of particular chemical or biochemical products. Several traditional metabolic engineering methods are commonly used to increase the production of a desired target, but the products are always far below their theoretical maximums. Using numeral optimisation algorithms to identify gene knockouts may stall at a local minimum in a multivariable function. This paper proposes a hybrid of the artificial bee colony (ABC) algorithm and the minimisation of metabolic adjustment (MOMA) to predict an optimal set of solutions in order to optimise the production rate of succinate and lactate. The dataset used in this work was from the iJO1366 Escherichia coli metabolic network. The experimental results include the production rate, growth rate and a list of knockout genes. From the comparative analysis, ABCMOMA produced better results compared to previous works, showing potential for solving genetic engineering problems.