This study analyzes and compares the results of leachate composition at the semi-aerobic Pulau Burung Landfill Site (PBLS) (unaerated pond and intermittently aerated pond) and the anaerobic Kulim Sanitary Landfill in the northern region of Malaysia. The raw samples were collected and analyzed for twenty parameters. The average values of the parameters such as phenols (1.2, 6.7, and 2.6 mg/L), total nitrogen (448, 1200, and 300 mg/L N-TN), ammonia-N (542, 1568, and 538 mg/L NH(3)-N), nitrite (91, 49, and 52 mg/L NO(2)(-)-N), total phosphorus (21, 17, and 19 mg/L), BOD(5) (83, 243, and 326 mg/L), COD (935, 2345, and 1892 mg/L), BOD(5)/COD (0.096,0.1124,0.205%), pH (8.20, 8.28, and 7.76), turbidity (1546, 180, and 1936 Formazin attenuation units (FAU)), and color (3334, 3347, and 4041 Pt Co) for leachate at the semi-aerobic PBLS (unaerated and intermittently aerated) and the anaerobic Kulim Sanitary Landfill were recorded, respectively. The obtained results were compared with previously published data and data from the Malaysia Environmental Quality Act 1974. The results indicated that Pulau Burung leachate was more stabilized compared with Kulim leachate. Furthermore, the aeration process in PBLS has a considerable effect on reducing the concentration of several pollutants. The studied leachate requires treatment to minimize the pollutants to an acceptable level prior to discharge into water courses.
A diesel-degrading bacterium has been isolated from a diesel-polluted site. The isolate was tentatively identified as Staphylococcus aureus strain DRY11 based on partial 16S rDNA molecular phylogeny and Biolog GP microplate panels and Microlog database. Isolate 11 showed an almost linear increase in cellular growth with respect to diesel concentrations with optimum growth occurring at 4% (v/v) diesel concentration. Optimization studies using different nitrogen sources showed that the best nitrogen source was potassium nitrite. Sodium nitrite was optimum at 1.2 g l(-1) and higher concentrations were strongly inhibitory to cellular growth. The optimal pH that supported growth of the bacterium was between 7.5 to 8.0 and the isolate exhibited optimal broad temperature supporting growth on diesel from 27 to 37 degrees C. An almost complete removal of diesel components was seen from the reduction in hydrocarbon peaks observed using Solid Phase Microextraction Gas Chromatography analysis after 5 days of incubation. The characteristics of this bacterium suggest that it is suitable for bioremediation of diesel spills and pollutions in the tropics.
Several local acrylamide-degrading bacteria have been isolated. One of the isolate that exhibited the highest growth on acrylamide as a nitrogen source was then further characterized. The isolate was tentatively identified as Bacillus cereus strain DRY135 based on carbon utilization profiles using Biolog GP plates and partial 16S rDNA molecular phylogeny. The isolate grew optimally in between the temperatures of 25 and 30 degrees C and within the pH range of 6.8 to 7.0. Glucose, fructose, lactose, maltose, mannitol, citric acid and sucrose supported growth with glucose being the best carbon source. Different concentrations of acrylamide ranging from 100 to 4000 mg l(-1) incorporated into the growth media shows that the highest growth was obtained at acrylamide concentrations of between 500 to 1500 mg l(-1). At 1000 mg l(-1) of acrylamide, degradation was 90% completed after ten days of incubation with concomitant cell growth. The metabolite acrylic acid was detected in the media during degradation. Other amides such as methacrylamide, nicotinamide, acetamide, propionamide and urea supported growth with the highest growth supported by acetamide, propionamide and urea. Strain DRY135, however was not able to assimilate 2-chloroacetamide. The characteristics of this isolate suggest that it would be useful in the bioremediation of acrylamide.
A novel microextraction technique termed solid phase membrane tip extraction (SPMTE) was developed. Selected triazine herbicides were employed as model compounds to evaluate the extraction performance and multiwall carbon nanotubes (MWCNTs) were used as the adsorbent enclosed in SPMTE device. The SPMTE procedure was performed in semi-automated dynamic mode and several important extraction parameters were comprehensively optimized. Under the optimum extraction conditions, the method showed good linearity in the range of 1-100 microg/L, acceptable reproducibility (RSD 6-8%, n=5), low limits of detection (0.2-0.5 microg/L), and satisfactory relative recoveries (95-101%). The SPMTE device could be regenerated and reused up to 15 analyses with no analyte carry-over effects observed. Comparison was made with commercially available solid phase extraction-molecular imprinted polymer cartridge (SPE-MIP) for triazine herbicides as the reference method. The new developed method showed comparable or even better results against reference method and is a simple, feasible, and cost effective microextraction technique.
Research was conducted to manufacture and evaluate a restructured turkey breast product using the Fibrimex cold-set binding system, sodium diacetate (NaD), and sodium lactate (NaL) and to ascertain effects of the treatments on proximate composition, pH, psychrotrophic organisms, water activity, onset of rancidity (TBA), thaw loss, cooking yields, and objective color, and sensory characteristics. Whole turkey breasts were cut into 5-cm-thick strips; treated with either water only (control), 1.5% NaL, 2.0% NaL, 0.1% NaD, 1.5% NaL + 0.1% NaD, or 2.0% NaL + 0.1% NaD; blended with Fibrimex ingredients; stuffed into casings; and stored at -30 degrees C for 0, 1, 2, and 3 mo. After each storage period, frozen chubs were tempered at 4 degrees C, sliced into 1-cm-thick steaks, packaged in retail trays, stored at 0 degrees C to simulate retail storage, and analyzed after 0, 2, 4, 6, 8, and 10 d. Sodium diacetate used alone or in combination with NaL reduced (P < 0.05) growth of psychrotrophic organisms and had no adverse effects on water activity, pH, cooking yield, fat, moisture, protein, objective color, onset of rancidity, and sensory characteristics (juiciness, turkey flavor intensity, and tenderness). Panelists reported slight off-flavor in all steaks treated with NaL. Treating steaks with NaL alone or in combination with NaD resulted in increased (P < 0.05) ash content. Sodium lactate also functioned to minimize thaw loss in the frozen restructured turkey product.
Oil palm trunk fibre (OPTF)--an agricultural solid waste--was used as low-cost adsorbent to remove malachite green (MG) from aqueous solutions. The operating variables studied were contact time, initial dye concentration, and solution pH. Equilibrium adsorption data were analyzed by three isotherms, namely the Freundlich isotherm, the Langmuir isotherm, and the multilayer adsorption isotherm. The best fit to the data was obtained with the multilayer adsorption. The monolayer adsorption capacity of OPTF was found to be 149.35 mg/g at 30 degrees C. Adsorption kinetic data were modeled using the Lagergren pseudo-first-order, Ho's pseudo-second-order and Elovich models. It was found that the Lagergren's model could be used for the prediction of the system's kinetics. The overall rate of dye uptake was found to be controlled by external mass transfer at the beginning of adsorption, then for initial MG concentrations of 25, 50, 100, 150, and 300 mg/L the rate-control changed to intraparticle diffusion at a later stage, but for initial MG concentrations 200 and 250 mg/L no evidence was found of intraparticle diffusion at any period of adsorption. It was found that with increasing the initial concentration of MG, the pore-diffusion coefficient increased while the film-diffusion coefficient decreased.
Direct recovery of hepatitis B core antigen (HBcAg) from unclarified Escherichia coli homogenates via expanded bed adsorption chromatography (EBA) has been explored in this study. Streamline DEAE was selected as the anion exchanger to recover HBcAg from heat-treated and non-heat-treated unclarified feedstocks. The use of anion-exchanger for direct extraction of proteins from unclarified feedstock is not preferred due to lack of specificity of its ligand. In this study, thermal treatment of the unclarified feedstock at 60 degrees C has resulted in 1.2- and 1.8-fold increases in yield and purity of HBcAg, respectively, compared with that purified from non-heat-treated feedstock. Heating the crude feedstock has resulted in denaturation and precipitation of contaminants in the feedstock, hence reducing non-specific interactions between the cell debris and adsorbent. The selectivity of the anion-exchanger has also been increased as shown in the breakthrough curve obtained. Enzyme-linked immunosorbent assay showed that the antigenicity of the HBcAg from heat-treated unclarified feedstock is still preserved.
A new sample pre-treatment technique termed cone-shaped membrane liquid phase microextraction (CSM-LPME) was developed and combined with micro-liquid chromatography (micro-LC) for the determination of selected pesticides in water samples. Four pesticides (hexaconazole, procymidone, quinalphos and vinclozolin) were considered as target analytes. Several important extraction parameters such as types of extraction solvent, agitation rate, pH value, total exposure time and effect of salt and humic acids were optimized. Enrichment factors of > 50 folds were easily achieved within 20 min of extraction. The analytical data demonstrated relative standard deviations for the reproducibility of the optimized CSM-LPME method ranging from 6.3 to 7.5%. The correlation coefficients of the calibration curves were at least 0.9995 across a concentration range of 2-100 microg/L. The detection limits for all the analytes were found to be in the range of 1.1-1.9 microg/L.
The nanobiocatalyst (NBC) is an emerging innovation that synergistically integrates advanced nanotechnology with biotechnology and promises exciting advantages for improving enzyme activity, stability, capability and engineering performances in bioprocessing applications. NBCs are fabricated by immobilizing enzymes with functional nanomaterials as enzyme carriers or containers. In this paper, we review the recent developments of novel nanocarriers/nanocontainers with advanced hierarchical porous structures for retaining enzymes, such as nanofibres (NFs), mesoporous nanocarriers and nanocages. Strategies for immobilizing enzymes onto nanocarriers made from polymers, silicas, carbons and metals by physical adsorption, covalent binding, cross-linking or specific ligand spacers are discussed. The resulting NBCs are critically evaluated in terms of their bioprocessing performances. Excellent performances are demonstrated through enhanced NBC catalytic activity and stability due to conformational changes upon immobilization and localized nanoenvironments, and NBC reutilization by assembling magnetic nanoparticles into NBCs to defray the high operational costs associated with enzyme production and nanocarrier synthesis. We also highlight several challenges associated with the NBC-driven bioprocess applications, including the maturation of large-scale nanocarrier synthesis, design and development of bioreactors to accommodate NBCs, and long-term operations of NBCs. We suggest these challenges are to be addressed through joint collaboration of chemists, engineers and material scientists. Finally, we have demonstrated the great potential of NBCs in manufacturing bioprocesses in the near future through successful laboratory trials of NBCs in carbohydrate hydrolysis, biofuel production and biotransformation.
A study was conducted to determine the total microbial population, the occurrence of growth promoting bacteria and their beneficial traits in acid sulfate soils. The mechanisms by which the bacteria enhance rice seedlings grown under high Al and low pH stress were investigated. Soils and rice root samples were randomly collected from four sites in the study area (Kelantan, Malaysia). The topsoil pH and exchangeable Al ranged from 3.3 to 4.7 and 1.24 to 4.25 cmol(c) kg(-1), respectively, which are considered unsuitable for rice production. Total bacterial and actinomycetes population in the acidic soils were found to be higher than fungal populations. A total of 21 phosphate-solubilizing bacteria (PSB) including 19 N2-fixing strains were isolated from the acid sulfate soil. Using 16S rRNA gene sequence analysis, three potential PSB strains based on their beneficial characteristics were identified (Burkholderia thailandensis, Sphingomonas pituitosa and Burkholderia seminalis). The isolated strains were capable of producing indoleacetic acid (IAA) and organic acids that were able to reduce Al availability via a chelation process. These PSB isolates solubilized P (43.65%) existing in the growth media within 72 hours of incubation. Seedling of rice variety, MR 219, grown at pH 4, and with different concentrations of Al (0, 50 and 100 µM) was inoculated with these PSB strains. Results showed that the bacteria increased the pH with a concomitant reduction in Al concentration, which translated into better rice growth. The improved root volume and seedling dry weight of the inoculated plants indicated the potential of these isolates to be used in a bio-fertilizer formulation for rice cultivation on acid sulfate soils.
The thermoalkaline protease enzyme from pitaya (Hylocereus polyrhizus) waste was purified by a factor of 221.2 with 71.3% recovery using ammonium sulphate precipitation, gel filtration, and cation exchange chromatography. Gel filtration chromatography together with sodium dodecyl sulphate gel electrophoresis (SDS-PAGE) revealed that the enzyme is monomeric with a molecular weight of 26.7 kDa. The apparent K m and V max of the protease were 2.8 mg/mL and 31.20 u/min, respectively. The optimum pH and temperature were 8.0 and 70°C. The enzyme was highly active and stable over a wide pH range (from pH 3.0 to pH 11.0 with the optimum activity at pH 8.0). The protease has broad specificity toward azocasein, casein, hemoglobin, and gelatine. Activity of the enzyme was inhibited by Fe(2+) and Zn(2+), while protease activity was increased in the presence of Ca(2+) and Mg(2+) and Cu(2+) by factors of 125%, 110%, and 105%, respectively. The alkaline protease showed extreme stability toward surfactants and oxidizing agent. The purified protease exhibited extreme stability in the presence of organic solvents and inhibitors. In addition, the enzyme was relativity stable toward organic solvents and chelating agents, such as ethylenediaminetetraacetic acid (EDTA). The enzyme, derived from pitaya peel, possesses unique characteristics and could be used in various industrial and biotechnological applications.
Stimuli-responsive bacterial cellulose-g-poly(acrylic acid) hydrogels were investigated for their potential use as an oral delivery system for proteins. These hydrogels were synthesized using electron beam irradiation without any cross-linking agents, thereby eliminating any potential toxic effects associated with cross-linkers. Bovine serum albumin (BSA), a model protein drug, was loaded into the hydrogels, and the release profile in simulated gastrointestinal fluids was investigated. Cumulative release of less than 10% in simulated gastric fluid (SGF) demonstrated the potential of these hydrogels to protect BSA from the acidic environment of the stomach. Subsequent conformational stability analyses of released BSA by SDS-PAGE, circular dichroism, and an esterase activity assay indicated that the structural integrity and bioactivity of BSA was maintained and preserved by the hydrogels. Furthermore, an increase in BSA penetration across intestinal mucosa tissue was observed in an ex vivo penetration experiment. Our fabricated hydrogels exhibited excellent cytocompatibility and showed no sign of toxicity, indicating the safety of these hydrogels for in vivo applications.
A maltogenic amylase (MAG1) from alkaliphilic Bacillus lehensis G1 was cloned, expressed in Escherichia coli, purified and characterised for its hydrolysis and transglycosylation properties. The enzyme exhibited high stability at pH values from 7.0 to 10.0. The hydrolysis of β-cyclodextrin (β-CD) produced malto-oligosaccharides of various lengths. In addition to hydrolysis, MAG1 also demonstrated transglycosylation activity for the synthesis of longer malto-oligosaccharides. The thermodynamic equilibrium of the multiple reactions was shifted towards synthesis when the reaction conditions were optimised and the water activity was suppressed, which resulted in a yield of 38% transglycosylation products consisting of malto-oligosaccharides of various lengths. Thin layer chromatography and high-performance liquid chromatography analyses revealed the presence of malto-oligosaccharides with a higher degree of polymerisation than maltoheptaose, which has never been reported for other maltogenic amylases. The addition of organic solvents into the reaction further suppressed the water activity. The increase in the transglycosylation-to-hydrolysis ratio from 1.29 to 2.15 and the increased specificity toward maltopentaose production demonstrated the enhanced synthetic property of the enzyme. The high transglycosylation activity of maltogenic amylase offers a great advantage for synthesising malto-oligosaccharides and rare carbohydrates.
Successful application of a magnetophoretic separation technique for harvesting biological cells often relies on the need to tag the cells with magnetic nanoparticles. This study investigates the underlying principle behind the attachment of iron oxide nanoparticles (IONPs) onto microalgal cells, Chlorella sp. and Nannochloropsis sp., in both freshwater and seawater, by taking into account the contributions of various colloidal forces involved. The complex interplay between van der Waals (vdW), electrostatic (ES) and Lewis acid-base interactions (AB) in dictating IONP attachment was studied under the framework of extended Derjaguin-Landau-Verwey-Overbeek (XDLVO) analysis. Our results showed that ES interaction plays an important role in determining the net interaction between the Chlorella sp. cells and IONPs in freshwater, while the AB and vdW interactions play a more dominant role in dictating the net particle-to-cell interaction in high ionic strength media (≥100 mM NaCl), such as seawater. XDLVO predicted effective attachment between cells and surface functionalized IONPs (SF-IONPs) with an estimated secondary minimum of -3.12 kT in freshwater. This prediction is in accordance with the experimental observation in which 98.89% of cells can be magnetophoretically separated from freshwater with SF-IONPs. We have observed successful magnetophoretic separation of microalgal cells from freshwater and/or seawater for all the cases as long as XDLVO analysis predicts particle attachment. For both the conditions, no pH adjustment is required for particle-to-cell attachment.
Xylose reductase (XR) is an oxidoreductase having potential applications in the production of various specialty products, mainly xylitol. It is important to screen for compounds that can decrease XR activity and consequently can decrease xylitol production. We have identified the byproducts in the hemicellulosic hydrolysate that inhibit XR from Candida tropicalis and measured their effects. XR inhibitory activities of byproducts, glucose, acetic acid, arabinose, lignin-degradation products (LDPs), furfural and hydroxymethylfurfural (HMF), were evaluated by measuring the MIC and IC50 values. XR activity was 11.2 U/ml. Acetic acid, LDPs, furfural and HMF significantly inhibited XR with IC50 values of 11, 6.4, 2.3 and 0.4 g/l, respectively. This is the first report on the inhibitory activities of several byproducts for XR.
Extraction of protease from a local ginger rhizome (Zingiber officinale var. Bentong) was carried out. The effect of extraction pH (6.4, 6.8, 7.0, 7.2, 7.6, 8.0, 8.4, and 8.8) and stabilizers (0.2% ascorbic acid, 0.2% ascorbic acid and 5 mM EDTA, or 10 mM cysteine and 5 mM EDTA) on protease activity during extraction was examined. pH 7.0 potassium phosphate buffer and 10 mM cysteine in combination with 5 mM EDTA as stabilizer were found to be the most effective conditions. The extraction procedure yielded 0.73% of Bentong ginger protease (BGP) with a specific activity of 24.8±0.2 U/mg protein. Inhibitory tests with some protease inhibitors classified the enzyme as a cysteine protease. The protease showed optimum activity at 60 °C and pH 6-8, respectively. The enzyme was completely inhibited by heavy metal cations such as Cu2+, and Hg2+. SDS stimulated the activity of enzyme, while emulsifiers (Tween 80 and Tween 20) slightly reduced its activity. The kinetic analysis showed that the protease has Km and Vmax values of 0.21 mg mL-1 and 34.48 mg mL-1 min-1, respectively. The dried enzyme retained its activity for 22 months when stored at -20 °C.
Xylose-rich sawdust hydrolysate can be an economic substrate for the enzymatic production of xylitol, a specialty product. It is important to identify the process factors influencing xylitol production. This research aimed to screen the parameters significantly affecting bioxylitol synthesis from wood sawdust by xylose reductase (XR). Enzymatic bioxylitol production was conducted to estimate the effect of different variables reaction time (2-18 h), temperature (20-70 °C), pH (4.0-9.0), NADPH (1.17-5.32 g/L), and enzyme concentration (2-6 %) on the yield of xylitol. Fractional factorial design was followed to identify the key process factors. The screening design identified that time, temperature, and pH are the most significant factors influencing bioxylitol production among the variables with the values of 12 h, 35 °C, and 7.0, respectively. These conditions led to a xylitol yield of 71 % (w/w). This is the first report on the statistical screening of process variables influencing enzyme-based bioxylitol production from lignocellulosic biomass.
Dominant strains of lactic acid bacteria (LAB) isolated from honey bees were evaluated for their γ-aminobutyric acid (GABA)-producing ability. Out of 24 strains, strain Taj-Apis362 showed the highest GABA-producing ability (1.76 mM) in MRS broth containing 50 mM initial glutamic acid cultured for 60 h. Effects of fermentation parameters, including initial glutamic acid level, culture temperature, initial pH and incubation time on GABA production were investigated via a single parameter optimization strategy. The optimal fermentation condition for GABA production was modeled using response surface methodology (RSM). The results showed that the culture temperature was the most significant factor for GABA production. The optimum conditions for maximum GABA production by Lactobacillus plantarum Taj-Apis362 were an initial glutamic acid concentration of 497.97 mM, culture temperature of 36 °C, initial pH of 5.31 and incubation time of 60 h, which produced 7.15 mM of GABA. The value is comparable with the predicted value of 7.21 mM.
Rice straw/magnetic nanocomposites (RS/Fe3O4-NCs) were prepared via co-precipitation method for removal of Pb(II) and Cu(II) from aqueous solutions. Response surface methodology (RSM) was utilized to find the optimum conditions for removal of ions. The effects of three independent variables including initial ion concentration, removal time, and adsorbent dosage were investigated on the maximum adsorption of Pb (II) and Cu (II). The optimum conditions for the adsorption of Pb(II) and Cu(II) were obtained (100 and 60 mg/L) of initial ion concentration, (41.96 and 59.35 s) of removal time and 0.13 g of adsorbent for both ions, respectively. The maximum removal efficiencies of Pb(II) and Cu(II) were obtained 96.25% and 75.54%, respectively. In the equilibrium isotherm study, the adsorption data fitted well with the Langmuir isotherm model. The adsorption kinetics was best depicted by the pseudo-second order model. Desorption experiments showed adsorbent can be reused successfully for three adsorption-desorption cycles.
Gamma-aminobutyric acid (GABA) is an important bioactive compound biosynthesized by microorganisms through decarboxylation of glutamate by glutamate decarboxylase (GAD). In this study, a full-length GAD gene was obtained by cloning the template deoxyribonucleic acid to pTZ57R/T vector. The open reading frame of the GAD gene showed the cloned gene was composed of 1410 nucleotides and encoded a 469 amino acids protein. To improve the GABA-production, the GAD gene was cloned into pMG36e-LbGAD, and then expressed in Lactobacillus plantarum Taj-Apis362 cells. The overexpression was confirmed by SDS-PAGE and GAD activity, showing a 53 KDa protein with the enzyme activity increased by sevenfold compared with the original GAD activity. The optimal fermentation conditions for GABA production established using response surface methodology were at glutamic acid concentration of 497.973 mM, temperature 36°C, pH 5.31 and time 60 h. Under the conditions, maximum GABA concentration obtained (11.09 mM) was comparable with the predicted value by the model at 11.23 mM. To our knowledge, this is the first report of successful cloning (clone-back) and overexpression of the LbGAD gene from L. plantarum to L. plantarum cells. The recombinant Lactobacillus could be used as a starter culture for direct incorporation into a food system during fermentation for production of GABA-rich products.