The biodegradation characteristics of palm oil mill effluent (POME) and the related microbial community were studied in both actual sequential anaerobic ponds in Malaysia and enrichment cultures. The significant degradation of the POME was observed in the second pond, in which the temperature was 35-37 °C. In this pond, biodegradation of major long chain fatty acids (LCFA), such as palmitic acid (C16:0) and oleic acid (C18:1), was also confirmed. The enrichment culture experiment was conducted with different feeding substrates, i.e. POME, C16:0 and C18:1, at 35 °C. Good recovery of methane indicated biodegradation of feeds in the POME and C16:0 enrichments. The methane production rate of the C18:1 enrichment was slower than other substrates and inhibition of methanogenesis was frequently observed. Denaturing gradient gel electrophoresis (DGGE) analyses indicated the existence of LCFA-degrading bacteria, such as the genus Syntrophus and Syntorophomonas, in all enrichment cultures operated at 35 °C. Anaerobic degradation of the POME under mesophilic conditions was stably processed as compared with thermophilic conditions.
The objective of this research was to study the kinetics of synthesis of a commercially important ester - Isopropyl Palmitate (IPP) using immobilized lipase (Lipozyme IM). It was studied in a packed bed differential reactor. In order to establish the kinetics of the reaction, parameters such as linear velocity of the fluid through the reactor, particle size, substrate concentration, substrate molar ratio, temperature and water activity were studied. Operational and storage stability of the enzyme were also assessed. The reaction followed Michaelis-Menton kinetics as observed from the relationship of initial rate of the reaction as a function of substrate concentration. It was found that the optimum substrate concentration was 0.15M palmitic acid and isopropyl alcohol in 1:1 stoichiometric ratio. Inhibition by excess of isopropyl alcohol has been identified. The optimum temperature for the esterification reaction was found to be around 50 degrees C. The activation energy of this process was determined to be 43.67 kJ/mol. The optimum water content was 0.50%. The reaction rates were measured in the absence of any significant external diffusional limitations. Since internal diffusional limitations could not be eliminated, the kinetics observed is only apparent.
Immobilized PS-C 'Amano' II lipase was used to catalyze the interesterification of palm olein (POo) with 30, 50, and 70% stearic acid in n-hexane at 60 degrees C. The catalytic performance of the immobilized lipase was evaluated by determining the composition change of fatty acyl groups and triacylglycerol (TAG) by gas liquid chromatography and high-performance liquid chromatography, respectively. The interesterification process resulted in the formation of new TAGs, mainly tripalmitin and dipalmitostearin, both of which were absent in the original oil. These changes in TAG composition resulted in an increase in slip melting point, from the original 25.5 degrees C to 36.3, 37.0, and 40.0 degrees C in the modified POo with 30, 50, and 70% stearic acid, respectively. All the reactions attained steady state in about 6 h. This type of work will find great applications in food industries, such as confectionery.
Bioprospecting for biodiesel potential in microalgae primarily involves a few model species of microalgae and rarely on non-model microalgae species. Therefore, the present study determined changes in physiology, oil accumulation, fatty acid composition and biodiesel properties of a non-model microalga Messastrum gracile SE-MC4 in response to 12 continuous days of nitrate-starve (NS) and nitrate-replete (NR) conditions respectively. Under NS, the highest oil content (57.9%) was achieved despite reductions in chlorophyll content, biomass productivity and lipid productivity. However, under both NS and NR, palmitic acid and oleic acid remained as dominant fatty acids thus suggesting high potential of M. gracile for biodiesel feedstock consideration. Biodiesel properties analysis returned high values of cetane number (CN 61.9-64.4) and degree of unsaturation (DU 45.3-57.4) in both treatments. The current findings show the possibility of a non-model microalga to inherit superior ability over model species in oil accumulation for biodiesel development.
Fatty acid desaturase enzymes are capable of inserting double bonds between carbon atoms of saturated fatty acyl-chains to produce unsaturated fatty acids. A gene coding for a putative Δ9-fatty acid desaturase-like protein was isolated from a cold-tolerant Pseudomonas sp. A8, cloned and heterologously expressed in Escherichia coli. The gene named as PA8FAD9 has an open reading frame of 1185 bp and codes for 394 amino acids with a predicted molecular weight of 45 kDa. The enzyme showed high Δ9-fatty acid desaturase-like protein activity and increased overall levels of cellular unsaturated fatty acids in the recombinant E. coli cells upon expression at different temperatures. The results showed that the ratio of palmitoleic to palmitic acid in the recombinant E. coli cells increased by more than twice the amount observed in the control cells at 20 °C using 0.4 mM IPTG. GCMS analysis confirmed the ability of this enzyme to convert exogenous stearic acid to oleic acid incorporated into the recombinant E. coli membrane phospholipids. It may be concluded that the PA8FAD9 gene from Pseudomonas sp. A8 codes for a putative Δ9-fatty acid desaturase protein actively expressed in E. coli under the influence of temperature and an inducer.
We recently developed a cell-penetrating drug carrier composed of albumin (HSA) combined with palmitoyl-cyclic-(D-Arg)12. While it is possible that the palmitoyl-cyclic-(D-Arg)12/HSA enters the cell mainly via macropinocytosis, the mechanism responsible for the induction of macropinocytosis and endosomal escape remain unknown. We report herein that palmitoyl-cyclic-(D-Arg)12/HSA might interact with heparan sulfate proteoglycan and the chemokine receptor CXCR4 followed by multiple activations of the PKC/PI3K/JNK/mTOR signaling pathways to induce macropinocytosis. This result was further confirmed by a co-treatment with 70 kDa dextran, a macropinocytosis marker. Using liposomes that mimic endosomes, the leakage of 5,6-carboxyfluorescein from liposome was observed in the presence of palmitoyl-cyclic-(D-Arg)12/HSA only in the case of the anionic late endosome-like liposomes but not the neutral early endosome-like liposomes. Heparin largely inhibited this leakage, suggesting the importance of electrostatic interactions between palmitoyl-cyclic-(D-Arg)12/HSA and the late-endosomal membrane. Immunofluorescence staining and Western blotting data indicated that the intact HSA could be transferred from endosomes to the cytosol. These collective data suggest that the palmitoyl-cyclic-(D-Arg)12/HSA is internalized via macropinocytosis and intact HSA is released from the late endosomes to the cytoplasm before the endosomes fuse with lysosomes. Palmitoyl-cyclic-(D-Arg)12/HSA not only functions as an intracellular drug delivery carrier but also as an inducer of macropinocytosis.
In this study, the effects of limited and excess nitrate on biomass, lipid production, and fatty acid profile in Messastrum gracile SE-MC4 were determined. The expression of fatty acid desaturase genes, namely stearoyl-ACP desaturase (SAD), omega-6 fatty acid desaturase (ω-6 FAD), omega-3 fatty acid desaturase isoform 1 (ω-3 FADi1), and omega-3 fatty acid desaturase isoform 2 (ω-3 FADi2) was also assessed. It was found that nitrate limitation generally increased the total oil, α-linolenic acid (C18:3n3) and total polyunsaturated fatty acid (PUFA) contents in M. gracile. The reduction of nitrate concentration from 1.76 to 0.11 mM increased the total oil content significantly from 32.5 to 41.85% (dry weight). Palmitic (C16:0) and oleic (C18:1) acids as the predominant fatty acids in this microalgae constituted between 82 and 87% of the total oil content and were relatively consistent throughout all nitrate concentrations tested. The expression of SAD, ω-6 FAD, and ω-3 FADi2 genes increased under nitrate limitation, especially at 0.11 mM nitrate. The ω-3 FADi1 demonstrated a binary up-regulation pattern of expression under both nitrate-deficient (0.11 mM) and -excess (3.55 mM) conditions. Thus, findings from this study suggested that limited or excess nitrate could be used as part of a cultivation strategy to increase oil and PUFA content following media optimisation and more efficient culture methodology. Data obtained from the expression of desaturase genes would provide valuable insights into their roles under excess and limited nitrate conditions in M. gracile, potentially paving the way for future genetic modifications.
Oil spills generally cause worldwide concern due to their detrimental effects on the environment and the economy. An assortment of commercial systems has been developed to control these spills, including the use of agricultural wastes as sorbents. This work deals with raw and modified mangrove barks (Rhizophora apiculata), an industrial lignocellulosic waste, as a low cost adsorbent for oil-product-spill cleanup in the aquatic environment. Mangrove bark was modified using fatty acids (oleic acid and palmitic acid) to improve its adsorption capacity. The oil sorption capacity of the modified bark was studied and compared with that of the raw bark. Kinetic tests were conducted with a series of contact times. The influence of particle size, oil dosage, pH and temperature on oil sorption capacity was investigated. The results showed that oleic acid treated bark has a higher sorption capacity (2,860.00 ± 2.00 mg/g) than untreated bark for Tapis crude oil. A correlation between surface functional groups, morphology and surface area of the adsorbent was studied by Fourier transform infrared spectrum, field emission scanning electron microscopy images and Brunauer-Emmett-Teller analysis. Isotherm study was conducted using the Langmuir and Freundlich isotherm models. The result showed that adsorption of crude oil on treated mangrove bark could be best described by the Langmuir model.
In the present study, we investigated the effect of long-acyl chain SFA, namely palmitic acid (16:0) and stearic acid (18:0), at sn-1, 3 positions of TAG on obesity. Throughout the 15 weeks of the experimental period, C57BL/6 mice were fed diets fortified with cocoa butter, sal stearin (SAL), palm mid fraction (PMF) and high-oleic sunflower oil (HOS). The sn-1, 3 positions were varied by 16:0, 18:0 and 18:1, whilst the sn-2 position was preserved with 18:1. The HOS-enriched diet was found to lead to the highest fat deposition. This was in accordance with our previous postulation. Upon normalisation of total fat deposited with food intake to obtain the fat:feed ratio, interestingly, mice fed the SAL-enriched diet exhibited significantly lower visceral fat/feed and total fat/feed compared with those fed the PMF-enriched diet, despite their similarity in SFA-unsaturated fatty acid-SFA profile. That long-chain SFA at sn-1, 3 positions concomitantly with an unsaturated FA at the sn-2 position exert an obesity-reducing effect was further validated. The present study is the first of its kind to demonstrate that SFA of different chain lengths at sn-1, 3 positions exert profound effects on fat accretion.
A single line flow injection analysis (FIA) method that incorporated a preconcentrator column packed with C(18) particles and capacitively coupled contactless conductivity detector (C(4)D) was developed for the determination of free fatty acid (FFA) in vegetable oils. The carrier stream was methanol/1.5 mM sodium acetate (pH 8) 80:20 (v/v) at a flow rate of 1.0 mL min(-1). Calibration curve was well correlated (r(2)=0.9995) within the range of 1-200 mg L(-1) FFA (expressed as palmitic acid). Sampling rate of 40-60 h(-1) was achieved. Good agreement was found between the standard non-aqueous titrimetry method and the proposed method when applied to the determination of FFA in palm (crude, olein, and refined, bleached and deodorised) and other vegetable (soybean, rice bran, walnut, corn and olive) oils. The proposed method offers distinct advantages over the official method, especially in terms of simplicity, high sampling rate, economy of solvents and sample, offering considerable promise as a low cost automated system that needs minimum human intervention over long periods of time.
Lipid biosynthesis and fatty acids composition of oleaginous zygomycetes, namely Cunninghamella bainieri 2A1, cultured in media with excess or limited nitrogen were quantitatively determined at different times of culture growth. Accumulation of lipids occurred even when the activity of NAD(+)-ICDH (β-Nicotinamide adenine dinucleotide-isocitrate dehydrogenase) was still detectable in both media. In C. bainieri 2A1, under nitrogen limitation, the ratio of lipids was around 35%, whereas in nitrogen excess medium (feeding media supplemented with ammonium tartarate), the lipid ratio decreased. The amount of this decrease depended on the level of ammonium tartarate in the media. The main findings in this paper were that C. bainieri 2A1 has the ability to accumulate lipid although nitrogen concentration detected inside the media and that NAD-ICDH was active in all culture periods. These results proved that the strain C. bainieri 2A1 has an alternative behavior in lipid biosynthesis that differs from yeast. According to the old hypotheses, yeasts could not accumulate lipid more than 10% when nitrogen was detected inside the media. Nitrogen-limited and excess media both contained the same fatty acids (palmitic acid, stearic acid, olic acid, linoleic acid and γ-linolenic acid), but at different concentrations. The C:N ratio was also studied and showed no effects on total lipid accumulation, but a significant effect on γ-linolenic acid concentration.
The seeds of Terminalia catappa from Malaysia were analyzed for their physicochemical properties. The following values were obtained: moisture 6.23 ± 0.09 %, ash 3.78 ± 0.04 %, lipid 54.68 ± 0.14 %, protein 17.66 ± 0.13 %, total dietary fibre 9.97 ± 0.08 %, carbohydrate 7.68 ± 0.06 %, reducing sugar 1.36 ± 0.16 %, starch 1.22 ± 0.15 %, caloric value 593.48 ± 0.24 %. Studies were also conducted on amino acid profile and free fatty acid composition of the seed oil. Results revealed that glutamic acid was the major essential amino acid while methionine and lysine were the limiting amino acids. The major saturated fatty acid was palmitic acid, while the main unsaturated fatty acid was oleic acid followed by linoleic acid. In addition, the seed was rich in sucrose and had trace amount of glucose and fructose. Briefly, the seed was high in proteins and lipids which are beneficial to human.
Effect of supercritical CO2 extracted Nigella sativa L. seed extract (NE) on frying performance of sunflower oil and refined, bleached and deodorized (RBD) palm olein was investigated at concentrations of 1.2 % and 1.0 % respectively. Two frying systems containing 0 % N. sativa L. extract (Control) and 0.02 % butylated hydroxytoluene (BHT) were used for comparison. Physicochemical properties such as fatty acid composition (FAC), Peroxide Value (PV), Anisidine Value (AV), Totox Value (TV), Total Polar Content (TPC), C18:2/C16:0 ratio and viscosity of frying oils were determined during five consecutive days of frying. Results have shown that N. sativa L. extract was able to improve the oxidative stability of both frying oils during the frying process compared to control. The stabilizing effect of antioxidants were in the order of BHT > NE. RBD palm olein was found to be more stable than sunflower oil based on the ratio of linoleic acid (C18:2) to palmitic acid (C16:0) and fatty acid composition.
Chromobacterium violaceum (C. violaceum) is a Gram-negative, rod-shaped facultatively anaerobic bacterium implicated with recalcitrant human infections. Here, we evaluated the anti-QS and antibiofilm activities of ethyl acetate extracts of Passiflora edulis (P. edulis) on the likely inactivation of acyl-homoserine lactone (AHL)-regulated molecules in C. violaceum both by in vitro and in silico analyses. Our investigations showed that the sub-MIC levels were 2, 1, and 0.5 mg/mL, and the concentrations showed a marked reduction in violacein pigment production by 75.8, 64.6, and 35.2%. AHL quantification showed 72.5, 52.2, and 35.9% inhibitions, inhibitions of EPS production (72.8, 36.5, and 25.9%), and reductions in biofilm formation (90.7, 69.4, and 51.8%) as compared to a control. Light microscopy and CLSM analysis revealed dramatic reduction in the treated biofilm group as compared to the control. GC-MS analysis showed 20 major peaks whose chemical structures were docked as the CviR ligand. The highest docking score was observed for hexadecanoic acid, 2-hydroxy-1-(hydroxymethyl) ethyl ester bonds in the active site of CviR with a binding energy of -8.825 kcal/mol. Together, we found that hexadecanoic acid, 2-hydroxy-1-(hydroxymethyl) ethyl ester remarkably interacted with CviR to inhibit the QS system. Hence, we concluded that hexadecanoic acid, 2-hydroxy-1-(hydroxymethyl) ethyl ester of P. edulis could likely be evaluated for treating C. violaceum infections.
Membrane-bound fatty acid desaturases perform oxygenated desaturation reactions to insert double bonds within fatty acyl chains in regioselective and stereoselective manners. The Δ9-fatty acid desaturase strictly creates the first double bond between C9 and 10 positions of most saturated substrates. As the three-dimensional structures of the bacterial membrane fatty acid desaturases are not available, relevant information about the enzymes are derived from their amino acid sequences, site-directed mutagenesis and domain swapping in similar membrane-bound desaturases. The cold-tolerantPseudomonassp. AMS8 was found to produce high amount of monounsaturated fatty acids at low temperature. Subsequently, an active Δ9-fatty acid desaturase was isolated and functionally expressed inEscherichia coli. In this paper we report homology modeling and docking studies of a Δ9-fatty acid desaturase from a Cold-tolerantPseudomonassp. AMS8 for the first time to the best of our knowledge. Three dimensional structure of the enzyme was built using MODELLER version 9.18 using a suitable template. The protein model contained the three conserved-histidine residues typical for all membrane-bound desaturase catalytic activity. The structure was subjected to energy minimization and checked for correctness using Ramachandran plots and ERRAT, which showed a good quality model of 91.6 and 65.0%, respectively. The protein model was used to preform MD simulation and docking of palmitic acid using CHARMM36 force field in GROMACS Version 5 and Autodock tool Version 4.2, respectively. The docking simulation with the lowest binding energy, -6.8 kcal/mol had a number of residues in close contact with the docked palmitic acid namely, Ile26, Tyr95, Val179, Gly180, Pro64, Glu203, His34, His206, His71, Arg182, Thr85, Lys98 and His177. Interestingly, among the binding residues are His34, His71 and His206 from the first, second, and third conserved histidine motif, respectively, which constitute the active site of the enzyme. The results obtained are in compliance with thein vivoactivity of the Δ9-fatty acid desaturase on the membrane phospholipids.
Third generation biofuels, also known as microalgal biofuels, are promising alternatives to fossil fuels. One attractive option is microalgal biodiesel as a replacement for diesel fuel. Chlamydomonas sp. Tai-03 was previously optimized for maximal lipid production for biodiesel generation, achieving biomass growth and productivity of 3.48 ± 0.04 g/L and 0.43 ± 0.01 g/L/d, with lipid content and productivity of 28.6 ± 1.41% and 124.1 ± 7.57 mg/L/d. In this study, further optimization using 5% CO2 concentration and semi-batch operation with 25% medium replacement ratio, enhanced the biomass growth and productivity to 4.15 ± 0.12 g/L and 1.23 ± 0.02 g/L/d, with lipid content and productivity of 19.4 ± 2.0% and 239.6 ± 24.8 mg/L/d. The major fatty acid methyl esters (FAMEs) were palmitic acid (C16:0), oleic acid (C18:1), and linoleic acid (C18:2). These short-chain FAMEs combined with high growth make Chlamydomonas sp. Tai-03 a suitable candidate for biodiesel synthesis.
Polygonum minus is a plant rich with bioactive components that contribute to food, pharmaceutical, and perfume industries. However, high moisture content in fresh plants will allow
microbial activity that leads to the degradation of plant quality. This can be prevented by
drying the fresh plants to preserve the characteristics of their bioactive components. The
present work was conducted to determine the effect of different drying methods such as
air-drying, oven-drying (40 and 60°C), and freeze-drying on essential oil (EO) yield and
chemical compounds of P. minus roots. For comparison purposes, all samples were extracted
by maceration with n-hexane at room temperature. Then, the samples were analysed and
identified by using gas chromatography-mass spectrometry (GC-MS). The highest EO yield
extract was obtained from freeze-drying (4.15 ± 0.5), followed by air-drying (3.79 ± 0.19). EO
yield from oven-drying at 40 and 60°C was 3.4 ± 0.14 and 0.86 ± 0.04, respectively. Results
showed that by increasing the drying temperature, the EO yield would decrease and cause a
loss of major chemical compounds in the P. minus root. Air-drying was found to be the best
method in preserving the presence of important chemical compound in P. minus roots such as
β-caryophyllene (1.43%), pentadecane (4.34%), hexadecanoic acid (3.91%) and oleic acid
(3.97%).
Mycelium-bound lipase (MBL) was prepared using a strain of Geotrichum candidum isolated from local soil. At the time of maximum lipase activity (54 h), the mycelia to which the lipase was bound were harvested by filtration and centrifugation. Dry MBL was prepared by lyophilizing the mycelia obtained. The yield of MBL was 3.66 g/l with a protein content of 44.11 mg/g. The lipase activity and specific lipase activity were 22.59 and 510 U/g protein, respectively. The moisture content of the MBL was 3.85%. The activity of free (extracellular) lipase in the culture supernatant (after removal of mycelia) was less than 0.2 U/ml. The MBL showed selectivity for oleic acid over palmitic acid during hydrolysis of palm olein, indicating that the lipase from G. candidum displayed high substrate selectivity for unsaturated fatty acid containing a cis-9 double bond, even in crude form. This unique specificity of MBL could be a direct, simple and inexpensive way in the fats and oil industry for the selective hydrolysis or transesterification of cis-9 fatty acid residues in natural triacylglycerols.
Studies were conducted to assess the percutaneous absorption of the triacyglycerols (TAGs), tocols and carotenoids present in crude and refined palm oil. In vitro experiments using upright Franz diffusion cells were employed to investigate the permeability of these compounds across full thickness human skin and into the receptor solution. Cetrimide, a cationic surfactant was chosen to be used as a solubilising agent in the receptor phase with an optimum concentration of 3.0 mg/mL and was able to provide sink conditions throughout the permeation. TAGs, tocols and carotenoids all permeated human skin from crude palm oil (CPO), whereas only TAGs permeated when refined palm oil (RPO) was used. Of the TAGs, oleic acid-containing TAGs was preferentially absorbed despite palmitic acid being the most prevalent fatty acid (FA) in TAGs. Tocols in the form of α-T3 showed the highest permeation followed by γ-T3, α-T and the lowest permeation was observed for δ-T3. Carotenoids (α-carotene and β-carotene) also showed an appreciable amount of permeation from CPO.
The aim of this study was to evaluate on how heat treatments by microwave oven may affect the oxidative degradation of sunflower oil (SFO) and its blend with palm olein (Po). The blend was prepared in the volume ratio of 40:60 (Po: SFO, PSF). The samples were exposed to microwave heating at medium power setting, for different periods. In this study, refractive index, free fatty acid content, peroxide value, p-anisidine value, total oxidation (Tomx), specific extinction, viscosity, polymer content, polar compounds and food oil sensor value of the oils all increased, whereas iodine value and C 18:21C16:0 ratio decreased as microwave heating progressed. Microwave heating temperature increased with increasing heating time and longer heating times resulted in a greater degree of oil deterioration. The percentage of linoleic acid tended to decrease, whereas the percentage of palmitic acid increased. The effect of adding PO to SFO on the formation of free fatty acids and conjugated dienes during microwave treatment was not significant (p< 0.05). No significant differences in food oil sensor value was observed between SFO and PSF. Based on the most oxidative stability criteria, it can be concluded that the microwave heating caused the formation of comparatively lower amounts of oxidation products in PSF compared to SFO, indicating a lower extent of oxidative degradation of PSF.