Removal of free fatty acid (FFA) in crude palm oil (CPO) using potassium oxide/dolomite as an adsorbent: Optimization by Taguchi method.

In this study, potassium oxide supported on dolomite adsorbent was used as an adsorbent for free fatty acids (FFAs) treatment in crude palm oil (CPO). The characteristics of the adsorbent were determined by TGA, XRD, SEM, BET and TPD-CO2. Taguchi method was utilized for experimental design and optimum condition determination. There were four parameters and three levels involved in this study: time (30, 60, 90 min), stirring rate (300, 500, 700 rpm), adsorbent dosage (1, 3, 5 wt%) and K2O concentration (5, 10, 15 wt%). The adsorbent had a larger pore size, higher basic strength, and more basic sites in greater efficiency (63%) in FFAs removal from CPO. The optimum conditions were at 30 min time, 700 rpm stirring rate, 5 wt% adsorbent dosage and 15 wt% K2O concentration. Taguchi method simplified determination of experimental parameters and minimized the operating costs.

Bioaccumulation of zinc in edible tropical vegetables in Peninsular Malaysia and its human health risk assessment based on various ethnicities in Malaysia.

Along with the growing utilization of zinc (Zn) and Zn-containing nanoparticles in various industries, Zn ecotoxicological evaluation on human food supply is necessary even though Zn is generally considered safe and rarely concentrated ecotoxicologically. This study aimed to investigate the bioaccumulation of Zn in 18 species of vegetables (seven leafy, nine fruity vegetables and one species each of tuber and legume) collected from two farming sites in the west coast of Peninsular Malaysia. A human health risk assessment (HHRA) was also conducted. In addition to HHRA based on the general population, HHRA based on each major ethnic group of the Malaysian society was also determined considering that the food consumption pattern would definitely be varied across ethnicities and age groups (children and adults). The study results showed that Zn concentrations were significantly higher (p < 0.05) in leafy vegetables than in other types of vegetables. However, the target hazard quotient (THQ) values were all found to be < 1.0. Therefore, based on the Malaysian ethnicities and age groups with their respective vegetable consumption patterns, the results indicated insignificant noncarcinogenic human health risk of Zn via oral consumption of vegetables by the Malaysian population. As a metric of measurement of HHRA, a comparison of THQ values could yield previously unreported insights into HHRA differences among the compared populations. A comparison of THQ values among the consumer groups indicated higher HHR for Chinese Malaysians and children due to their higher vegetable consumption and lower body weight, respectively. A comparison the Zn intakes of all the consumer groups with the recommended nutrient intakes indicated that the oral consumption of the vegetable species collected in this study would not result in Zn-related hazards and would not be able to fulfil the Zn dietary need of the individual consumer.

Toxic heavy metal (Pb and Cd) content in tobacco cigarette brands in Selangor state, Peninsular Malaysia.

Cadmium (Cd) and lead (Pb) are ubiquitous metals widely distributed in the environment, resulting in toxic health effects. This project aims to evaluate Pb and Cd as toxic elements in 15 different tobacco cigarette brands produced and/or sold in Selangor state, Peninsular Malaysia. The concentrations of Pb and Cd in all tobacco brands tested in this study were determined using the air-acetylene flame atomic absorption spectrophotometer (AAS). On average, the concentrations of Pb and Cd in different tobacco brand samples ranged from 3.05 and 0.80 µg/g dw, respectively. The results indicate that assessment mean values of Pb inhaled from smoking one packet of 20 cigarettes were in the range of 1.55-3.51 µg. Furthermore, the concentration of Cd inhaled per packet of cigarettes (20 sticks) is estimated to be 0.04-0.36 µg. However, there was a significant difference in the concentrations of Pb and Cd between the different brands of tobacco cigarettes, among cigarette prices (cheap versus expensive) of tobacco brands. In conclusion, cigarette smokers in Selangor, Malaysia, are heavily exposed to Pb and Cd, and such exposure could adversely affect their health in the long term. The impact of toxic heavy metals on smokers in this state would be an area for future research.

Relationship between Pb and Cd accumulations in house crow, their habitat, and food content from Klang area, Peninsular Malaysia.

Heavy metal pollution has become a global concern due to accumulation in tissue and transferable effects to humans via the food chain. This study focused on monitoring the accumulation of cadmium (Cd) and lead (Pb) in surface soil and body content: bone, heart, brain, liver, lung, muscle, kidney, feathers, feces, and gizzard contents of house crow Corvus splendens in the Klang region, Malaysia. The results revealed the occurrence of Pb and Cd in all biological samples from house crows, food contents, and surface soil samples. Heart and kidney accrued high amounts of Cd, while high amounts of Pb were found to accumulate in bones and feathers. Major discrepancies were also discovered in the concentrations of metals between juvenile and adults, as well as female and male bird samples. Concentrations of Pb and Cd in house crow internal tissues correlated significantly with that of bird feathers, but none could be established with that of surface soil. In addition, a significant correlation was observed between Pb concentration in the internal tissues to that of the feces, but the same was not the case when compared with the surface soil concentration. Metal accrual in the house crows feathers and feces may be through a long-term transmission via the food chain, which are eliminated from feathers via molting. This may suggest the utility of molted breast feathers of house crow in the bio-monitoring of Cd and Pb contamination, whereas feces of house crow appear only to be suitable for the bio-monitoring of Pb contamination.

Exposure of the endangered Milky stork population to cadmium and lead via food and water intake in Kuala Gula Bird Sanctuary, Perak, Malaysia.

The Milky stork is listed as an endangered species endemic to the Southeast Asia region. In Malaysia, the population is currently being reintroduced back into the wild. However, the increase of anthropogenic activity throughout the coastal area might expose the population to hazardous chemicals such as heavy metals. This study highlights the contamination of cadmium (Cd) and lead (Pb) in the Milky stork's diet. Additionally, this is the first time an integrated exposure model being used to assess heavy metal exposure risk to the population. Lead level (5.5-7.98 mg kg-1) in particular was relatively high compared to Cd (0.08-0.33 mg kg-1). This was probably related to the different niches occupied by the species in the aquatic environment. The results further show that the predicted exposure doses (through intake of both food and water) for all metals are much lower than the Tolerable Daily Intake (TDI) values. The total exposure dose for Cd was 0.11 mg kg-1 d-1 with TDI value of 0.54 mg kg-1 d-1 while Pb total exposure dose was 0.31 mg kg-1 d-1 with TDI value of 0.64 mg kg-1 d-1. Several possible factors that could lead to the observed pattern were discussed. In conclusion, there is an urgent need to improve the current habitat quality to protect the endangered species. The authors also emphasized on the protection of remaining Milky stork's habitats i.e. mudflats and mangroves and the creation of buffer zone to mitigate the negative impacts that may arise from pollution activity.

Distribution, mobility, and pollution assessment of Cd, Cu, Ni, Pb, Zn, and Fe in intertidal surface sediments of Sg. Puloh mangrove estuary, Malaysia.

Sungai Puloh mangrove estuary supports a large diversity of macrobenthic organisms and provides social benefits to the local community. Recently, it became a major recipient of heavy metals originating from industries in the hinterland as a result of industrialization and urbanization. This study was conducted to evaluate mobility and pollution status of heavy metals (Cd, Cu, Ni, Pb, Zn, and Fe) in intertidal surface sediments of this area. Surface sediment samples were collected based on four different anthropogenic sources. Metals concentrations were analyzed using an atomic absorption spectrophotometer (AAS). Results revealed that the mean concentrations were Zn (1023.68 ± 762.93 µg/g), Pb (78.8 ± 49.61 µg/g), Cu (46.89 ± 43.79 µg/g), Ni (35.54 ± 10.75 µg/g), Cd (0.94 ± 0.29 µg/g), and Fe (7.14 ± 0.94%). Most of the mean values of analyzed metals were below both the interim sediment quality guidelines (ISQG-low and ISQG-high), except for Pb concentration (above ISQG-low) and Zn concentration (above ISQG-high), thus suggesting that Pb and Zn may pose some environmental concern. Cadmium, Pb, and Zn concentrations were above the threshold effect level (TEL), indicating seldom adverse effect of these metals on macrobenthic organisms. Pollution load index (PLI) indicated deterioration and other indices revealed the intertidal surface sediment is moderately polluted with Cd, Pb, and Zn. Therefore, this mangrove area requires urgent attention to mitigate further contamination. Finally, this study will contribute to data sources for Malaysia in establishing her own ISQG since it is a baseline study with detailed contamination assessment indices for surface sediment of intertidal mangrove area.

Efficiency of ligation-mediated PCR and TAIL-PCR methods for isolation of RbcS promoter sequences from green microalgae Ankistrodesmus convolutus.

Isolation of promoter sequences from known gene sequences is a tedious task in genome-related research. An efficient method of obtaining the promoter sequences is necessary in order to successfully use targeted promoters for genetic manipulations. Here, efficiency and usefulness of two PCR-based methods, namely: ligation-mediated PCR and thermal asymmetric interlaced (TAIL) PCR, for isolation of promoter sequences of the ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (RbcS) gene from green microalgae Ankistrodesmus convolutus (A. convolutus) were evaluated. The results showed that the amplification efficiency of TAIL-PCR was higher than that of the ligation-mediated PCR method, i.e. the amplified promoter fragments of 1.2 and 0.8 kb in length or promoter sequences of 813 and 606 bp (after eliminating the unreadable sequences). The use of TAIL-PCR described here presents a low cost and efficient strategy for the isolation of promoter sequences of known genes, especially in GC-rich regions, and species with little or no available genome information such as A. convolutus.

Sequence analysis and potentials of the native RbcS promoter in the development of an alternative eukaryotic expression system using green Microalga Ankistrodesmus convolutus.

The availability of highly active homologous promoters is critical in the development of a transformation system and improvement of the transformation efficiency. To facilitate transformation of green microalga Ankistrodesmus convolutus which is considered as a potential candidate for many biotechnological applications, a highly-expressed native promoter sequence of ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (AcRbcS) has been used to drive the expression of ß-glucuronidase (gusA) gene in this microalga. Besides the determination of the transcription start site by 5'-RACE, sequence analysis revealed that AcRbcS promoter contained consensus TATA-box and several putative cis-acting elements, including some representative light-regulatory elements (e.g., G-box, Sp1 motif and SORLIP2), which confer light responsiveness in plants, and several potential conserved motifs (e.g., CAGAC-motif, YCCYTGG-motifs and CACCACA-motif), which may be involved in light responsiveness of RbcS gene in green microalgae. Using AcRbcS promoter::gusA translational fusion, it was demonstrated that this promoter could function as a light-regulated promoter in transgenic A. convolutus, which suggested that the isolated AcRbcS promoter was a full and active promoter sequence that contained all cis-elements required for developmental and light-mediated control of gene expression, and this promoter can be used to drive the expression of heterologous genes in A. convolutus. This achievement therefore advances the development of A. convolutus as an alternative expression system for the production of recombinant proteins. This is the first report on development of gene manipulation system for unicellular green alga A. convolutus.

Studies on the genetic variation of the green unicellular alga Haematococcus pluvialis (Chlorophyceae) obtained from different geographical locations using ISSR and RAPD molecular marker.

Haematococcus pluvialis (Flotow) is a unicellular green alga, which is considered to be the best astaxanthin-producing organism. Molecular markers are suitable tools for the purpose of finding out genetic variations in organisms; however there have been no studies conducted on ISSR or RAPD molecular markers for this organism. The DNA of 10 different strains of H. pluvialis (four strains from Iran, two strains from Finland, one strain from Switzerland and three strains from the USA) was extracted. A genetic similarity study was carried out using 14 ISSR and 12 RAPD primers. Moreover, the molecular weights of the bands produced ranged from 0.14 to 3.4 Kb. The PCA and dendrogram clustered the H. pluvialis strains into various groups according to their geographical origin. The lowest genetic similarity was between the Iran2 and USA2 strains (0.08) and the highest genetic similarity was between Finland1 and Finland2 (0.64). The maximum numbers of bands produced by the ISSR and RAPD primers were 35 and 6 bands, respectively. The results showed that ISSR and RAPD markers are useful for genetic diversity studies of Haematococcus as they showed geographical discrimination.

Cloning and characterization of ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (RbcS) cDNA from green microalga Ankistrodesmus convolutus.

An initial study on gene cloning and characterization of unicellular green microalga Ankistrodesmus convolutus was carried out to isolate and characterize the full-length cDNA of ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (RbcS) as a first step towards elucidating the structure of A. convolutus RbcS gene. The full-length of A. convolutus RbcS cDNA (AcRbcS) contained 28 bp of 5' untranslated region (UTR), 225 bp of 3' non-coding region, and an open reading frame of 165 amino acids consisting of a chloroplast transit peptide with 24 amino acids and a mature protein of 141 amino acids. The amino acid sequence has high identity to those of other green algae RbcS genes. The AcRbcS contained a few conserved domains including protein kinase C phosphorylation site, tyrosine kinase phosphorylation site and N-myristoylation sites. The AcRbcS was successfully expressed in Escherichia coli and a ~21 kDa of anticipated protein band was observed on SDS-PAGE. From the phylogenetic analysis of RbcS protein sequences, it was found that the RbcS of A. convolutus has closer genetic relationship with green microalgae species compared to those of green seaweed and green macroalgae species. Southern hybridization analysis revealed that the AcRbcS is a member of a small multigene family comprising of two to six members in A. convolutus genome. Under different illumination conditions, RT-PCR analysis showed that AcRbcS transcription was reduced in the dark, and drastically recovered in the light condition. Results presented in this paper established a good foundation for further study on the photosynthetic process of A. convolutus and other green algae species where little information is known on Rubisco small subunit.

Construction of cDNA library and preliminary analysis of expressed sequence tags from green microalga Ankistrodesmus convolutus Corda.

Green microalga Ankistrodesmus convolutus Corda is a fast growing alga which produces appreciable amount of carotenoids and polyunsaturated fatty acids. To our knowledge, this is the first report on the construction of cDNA library and preliminary analysis of ESTs for this species. The titers of the primary and amplified cDNA libraries were 1.1×10(6) and 6.0×10(9) pfu/ml respectively. The percentage of recombinants was 97% in the primary library and a total of 337 out of 415 original cDNA clones selected randomly contained inserts ranging from 600 to 1,500 bps. A total of 201 individual ESTs with sizes ranging from 390 to 1,038 bps were then analyzed and the BLASTX score revealed that 35.8% of the sequences were classified as strong match, 38.3% as nominal and 25.9% as weak match. Among the ESTs with known putative function, 21.4% of them were found to be related to gene expression, 14.4% ESTs to photosynthesis, 10.9% ESTs to metabolism, 5.5% ESTs to miscellaneous, 2.0% to stress response, and the remaining 45.8% were classified as novel genes. Analysis of ESTs described in this paper can be an effective approach to isolate and characterize new genes from A. convolutus and thus the sequences obtained represented a significant contribution to the extensive database of sequences from green microalgae.

Rapid and effective method of RNA isolation from green microalga Ankistrodesmus convolutus.

The rapid and effective method for the isolation of RNA from green microalga Ankistrodesmus convolutus based on homogenization in a simple CTAB buffer and selective precipitation of RNA with lithium chloride is developed. This procedure avoids the use of toxic chaotropic agents and phenol while high concentration of dithiothreitol is used to inhibit RNase activity and prevent oxidative cross-linking of nucleic acids by phenolics. The extraction procedure was able to produce high quality and intact RNA from A. convolutus. The yield of total RNA was 0.69-0.73 mg/g of fresh weight, with A(260)/A(280) ratio of 1.79-1.86. The obtained RNA was of sufficient quality and suitable for downstream application such as RT-PCR and cDNA library construction. The procedure may also have wider applicability for total RNA isolation from other green microalgae species.