Long-chain Fatty Acid Methyl Esters Research Articles

Thermodynamically predicting liquid/solid phase change of long-chain fatty acid methyl esters (FAMEs) and its application in evaluating the low-temperature performance of biodiesel

BackgroundLong-chain FAMEs, also called biodiesel, are viewed as the alternative to petroleum diesel for renewability and sustainability. Because the fatty acid profiles significantly varied for the sources, the compositions significantly affected the biodiesel properties, such as cloud points. The cloud point of biodiesel indicates the phenomenon of solid-liquid phase change. Previous models for cloud point predictions were limited to known components in the mixtures. MethodsThe cloud points of the binary, ternary and multicomponent mixtures of fatty acid methyl esters were measured in this study. A cloud point prediction model was established based on phase equilibrium with the modified Universal functional activity coefficient (UNIFAC) model and excess fusion enthalpy for predicting the nonideal behaviors of the components resulting from the molecular shape and molecule interactions. Significant findingsThe developed thermodynamic model accurately predicts the cloud point according to compositions. The model extends the application scope to the low-temperature range. There had eutectic points in the phase diagrams when the mixtures consisted of either saturated FAMEs or unsaturated FAMEs. This study proved that saturated FAMEs regulate the cloud points, but unsaturated FAMEs affect them through group interactions. Moreover, the proposed model can predict unknown FAMEs mixture as it is built on the group contributions.

Developing an Extreme Learning Machine-Based Model for Estimating the Isothermal Compressibility of Biodiesel

Nowadays, the high consumption of fossil fuels has caused many pollutants and environmental problems. Biodiesel has recently been considered as a clean and renewable alternative to fossil fuels. They are found in some molecular structures including fatty acid ethyl esters (FAEEs) and also fatty methyl esters (FAMEs), having various thermophysical characteristics. Thus, it appears essential to select the suitable methods for a particular diesel engine to estimate the ester characteristics. The current research sets out to develop a new and robust method predicting isothermal compressibility of long-chain fatty acid methyl and ethyl esters directly from several basic efficient parameters (pressure, temperature, normal melting point, and molecular weight). Therefore, as a novel and prevailing mathematical method in this field, an extreme learning machine was implemented for isothermal compressibility on the massive dataset. According to statistical evaluations, this novel established model had high accuracy and applicability (R2 = 1 and RMSE = 0.0018714) which is more accurate than previous models presented by former researchers. Among various factors of the sensitivity analysis, temperature and pressure had the greatest effect on the output values, so that the output parameter has a direct relationship with temperature and an inverse relationship with pressure with relevancy factors of 22.44% and −79.81%.

The Reservoir Age Effect Varies With the Mobilization of Pre-Aged Organic Carbon in a High-Altitude Central Asian Catchment

Lake sediments provide excellent archives to study past environmental and hydrological changes at high temporal resolution. However, their utility is often restricted by chronological uncertainties due to the “reservoir age effect” (RAE), a phenomenon that results in anomalously old radiocarbon ages of total organic carbon (TOC) samples that is mainly attributed to the contribution of pre-aged carbon from aquatic organisms. Although the RAE is a well-known problem especially in high altitude lakes, detailed studies analyzing the temporal variations in the contribution of terrestrial and aquatic organic carbon (OC) on the RAE are scarce. This is partially due to the complexity of isolating individual compounds for subsequent compound-specific radiocarbon analysis (CSRA). We developed a rapid method for isolating individual short-chain (C16and C18) and long-chain (>C24) saturated fatty acid methyl esters (FAMEs) by using high-pressure liquid chromatography (HPLC). Our method introduces only minor contaminations (0.50 ± 0.22 µg dead carbon on average) and requires only few injections (≤10), therefore offering clear advantages over traditional preparative gas chromatography (prep-GC). Here we show that radiocarbon values (Δ14C) of long-chain FAs, which originate from terrestrial higher plant waxes, reflect carbon from a substantially pre-aged OC reservoir, whereas the Δ14C of short-chain FAs that originate from aquatic sources were generally less pre-aged.14C ages obtained from the long-chain FAs are in closer agreement with14C ages of the corresponding bulk TOC fraction, indicating a high control of pre-aged terrestrial OC input from the catchment on TOC-derived14C ages. Variations in the age offset between terrestrial and aquatic biomarkers are related to changes in bulk sediment log(Ti/K) that reflect variations in detrital input from the catchment. Our results indicate that the chronological offset between terrestrial and aquatic OC in this high-altitude catchment is mainly driven by temporal variations in the mobilization of pre-aged OC from the catchment. In conclusion, to obtain accurate and process-specific lake sediment chronologies, attention must be given to the temporal dynamics of the RAE. Variations in the apparent ages of aquatic and terrestrial contributions to the sediment and their mass balance can substantially alter the reservoir age effect.

The enhanced lipid productivity of Chlorella minutissima and Chlorella pyrenoidosa by carbon coupling nitrogen manipulation for biodiesel production.

Biodiesel production from microalgae has been researched extensively and attempted to commercialize on a large scale, but there are major hurdles in the production process like harvesting and low lipid content, which should be studied to enhance the process and make it economical. Present study aimed to improve the lipid productivity of Chlorella minutissima and Chlorella pyrenoidosa by modifying the carbon and nitrogen content of the medium. Both organisms were grown in BG11 medium for the first 6days and thereafter grown in a modified BG11 medium completely deprived of nitrogen for 2 to 10days. Nitrogen deprivation increased the lipid productivity of Chlorella minutissima to 20% and that of Chlorella pyrenoidosa to 17.6% by day 6. This was further coupled with carbon addition in the form of citric acid (5g/L), sodium acetate (5g/L), sodium carbonate (5g/L), and sodium potassium tartarate (5g/L), which increased the total lipid productivity of Chlorella minutissima up to 24% and that of Chlorella pyrenoidosa up to 23%. The highest lipid productivity of up to 24% for Chlorella minutissima and up to 23% for Chlorella pyrenoidosa was observed with nitrogen deprivation coupled with sodium acetate. Acidic transesterification revealed the presence of fatty acid methyl esters, majority of which consisted of hexadecanoic acid methyl ester and octadecanoic acid methyl ester. Maximum of 3% fatty acid methyl esters for Chlorella minutissima and 4% for Chlorella pyrenoidosa were obtained under nitrogen deprivation and sodium acetate as a carbon source. Thus, nitrogen deprivation coupled with sodium acetate as an increased carbon source in BG11 medium helps to increase the lipid productivity of Chlorella minutissima and Chlorella pyrenoidosa, and produces long-chain fatty acid methyl esters of C17 and C19 along with C21, C25, and C29.

Identification of very long-chain (>C24) fatty acid methyl esters using gas chromatography coupled to quadrupole/time-of-flight mass spectrometry with atmospheric pressure chemical ionization source

Gas chromatography coupled to a quadrupole time-of-flight mass analyzer (QTOF) with an atmospheric pressure chemical ionization (APCI) source has been tested to study the ionization and mass spectrometric behavior of long-chain and very long-chain polyunsaturated fatty acids (LC-PUFAs, C18–24; VLC-PUFAs, >C24). The protonated molecule ([M+H]+), measured at accurate mass, became the base peak of the spectrum for all the studied compounds and was promoted by the addition of water into the source. This fact overcame the existing difficulties for the identification of VLC-PUFAs when using an electron ionization source (EI). The extensive fragmentation of PUFAs in this source is the main drawback due to the fact that since reference standards are not commercially available, final identification relies on retention time estimation. The application of GC−APCI-QTOF to the screening of lipid extracts from the eyes of different fish species added confidence to the identification of several VLC-PUFAs. Further investigation of ion ratios allowed to predict the position of key double bonds enabling the classification of VLC-PUFAs as ω3 or ω6 compounds. VLC-PUFAs spectra found in biological samples were compared to those obtained from corresponding peaks found in heterologous expression experiments of fish's Elovl4.

GC/MS Analysis of Long-Chain Aldehydes from Recent Coral

The main objective of this study is to present the chromatograms and mass spectra of aldehydes so that geochemists could easily identify these compounds in sediment samples. Aldehydes are rare compounds found in the surface environment, particularly in sediments. To date, aldehydes have not been used as environmental proxies. In this study, long-chain aldehydes detected from coral collected from the east coast of Kenya are analyzed by gas chromatography mass spectrometry (GC/MS) using the typical procedure for biomarker analysis. The retention time of the long-chain aldehydes and fatty acid methyl esters slightly overlapped. Hence, the peak corresponding to the aldehyde in the GC chromatogram is buried with that corresponding to the ester, possibly overlooking the aldehyde. After assessing the difference in the retention time between the aldehydes and fatty acids methyl ester, the aldehydes were easily detected using the standard analytical procedure for biomarkers analysis. A molecular ion (M+) with greater than 23 carbon atoms was detected from long-chain aldehydes. The base peak was observed at m/z 82 in the mass spectra of the aldehyde, where characteristic 68 + 14n ions corresponding to even-numbered ions were observed. The M-18 ion, which was formed by the loss of H2O, as well as M-46 or M-84 ions, was characteristic ions observed for long-chain aldehydes of the number of all carbon. The results obtained from this work, in which aldehydes were identified from coral samples, represent a good example of what could be achieved by geochemists when working with sediment samples and using aldehydes as environmental proxies.

Isolation and identification of an extracellular enzyme from Aspergillus Niger with Deoxynivalenol biotransformation capability

In this study, the purification and characterization of an extracellular enzyme form Aspergillus niger was performed. With an optimized protocol, it was conducted a 42.6-fold purification with a yield of 26.2%. The purified lipase had a monomeric molecular weight of 40.5kDa and an isoelectric point of 6.01, and its maximum enzyme activity could be achieved at 40°C and pH 7.5-9.0. The enzyme could be activated by Ca2+, Mg2+ and Fe2+, while its activity could be inhibited by Zn2+ and Cu2+. Additionally, organic compounds exerted an inhibitory effect on the enzyme activity in a descending order of methanol, ethanol, DMSO, EDTA, acetone. Meanwhile, the specificity analysis of the enzyme indicated a preference to tributyrin and vegetable oils as well as long-chain fatty acid methyl esters (C12-C18). Most importantly, this enzyme could successfully transform deoxynivalenol (DON). Using HPLC analysis，it was detected a biotransformation rate of more than 70%.The liquid chromatography-mass spectrometry (LC-MS) analysis showed that the molecular weight of the transformation product was 18.0 larger than that of DON, indicating that DON could be hydrolyzed by the enzyme. Overall, the proposed method here provides a new avenue for reducing the toxicity of DON, which appears to have a wide application outlook for DON biotransformation.

Enhanced expression of lipase I from Galactomyces geotrichum by codon optimisation in Pichia pastoris

Relatively poor heterologous protein yields have limited the commerical applications of Galactomyces geotrichum lipase I (GGl I) efficacy trials. To address this, we have redesigned the GGl I gene to preferentially match codon frequencies of Pichia pastoris (P. pastoris) while retaining the same amino acid sequence. The wild type and codon optimised GGl I (GGl I-wt and GGl I-op) were synthesised and cloned into pPICZαA with an N-terminal 6 × His tag sequence and expressed in P. pastoris X 33. The hydrolytic activity of GGl I-op was 150 U/mL, whereas the activity of the GGl I-wt could not be detected. GGl I-op recombinant proteins were purified by Ni-affinity chromatography and then characterised. The identity and purity of GGl I were confirmed by SDS-PAGE, MALDI-TOF mass spectrometry and Western blot analysis. Enzymatic deglycosylation was used to show that the lipase is a glycosylated protein, containing ∼10% sugar. The molecular weight (MW) of the GGl I secreted by recombinant P. pastoris was approximated at 63 kDa. The optimum pH and temperature of the recombinant lipase were 8.0 and 35 °C, respectively. The enzyme was active over a broad pH range (7.0–9.0) and temperature range (20 °C–45 °C). The lipase showed high activity toward medium- and long-chain fatty acid methyl esters (C8-C16) and retained much of its activity in the presence of Tween-80 and Trition X-100. Lipase activity was stimulated by Mg2+, Ca2+, Mn2+ and Cu2+ and inhibited by Fe2+, Fe3+, Zn2+ and Co2+. This lipase may prove useful to the detergent industry and in organic synthesis reactions.

Development of an Agrobacterium-Mediated Transformation Method and Evaluation of Two Exogenous Constitutive Promoters in Oleaginous Yeast Lipomyces starkeyi

Oleaginous yeast Lipomyces starkeyi, a promising strain of great biotechnical importance, is able to accumulate over 60% of its cell biomass as triacylglycerols (TAGs). It is promising to directly produce the derivatives of TAGs, such as long-chain fatty acid methyl esters and alkanes, in L. starkeyi. However, techniques for genetic modification of this oleaginous yeast are lacking, thus, further research is needed to develop genetic tools and functional elements. Here, we used two exogenous promoters (pGPD and pPGK) from oleaginous yeast Rhodosporidium toruloides to establish a simpler Agrobacterium-mediated transformation (AMT) method for L. starkeyi. Hygromycin-resistant transformants were obtained on antibiotic-contained plate. Mitotic stability test, genotype verification by PCR, and protein expression confirmation all demonstrated the success of this method. Furthermore, the strength of these two promoters was evaluated at the phenotypic level on a hygromycin-gradient plate and at the transcriptional level by real-time quantitative PCR. The PGK promoter strength was 2.2-fold as that of GPD promoter to initiate the expression of the hygromycin-resistance gene. This study provided an easy and efficient genetic manipulation method and elements of the oleaginous yeast L. starkeyi for constructing superior strains to produce advanced biofuels.

Relative Reactivity of Oxygenated Fuels: Alcohols, Aldehydes, Ketones, and Methyl Esters

This work aims at comparing and highlighting the main reaction pathways characterizing the combustion behavior of oxygenated fuels. Ethanol and heavier alcohols are already viable biofuels, despite some concern about their aldehydes and ketones emissions. Recently, the potential of 2-butanone (methyl ethyl ketone, MEK) as anti-knocking fuel was investigated at engine-relevant conditions. Moving from methyl butanoate (MB), long-chain fatty acid methyl esters are largely considered and used as biodiesels, mainly in Europe. Starting from a consistent assessment of C–H and C–C bond dissociation energies (BDEs) in n-butane, n-butanol, n-butanal, MEK, and MB, their impact on the selectivity of the different H-abstraction reactions and their relative reactivity are analyzed. Low-temperature oxidation mechanisms of 1-butanol and 2-butanone are also presented and discussed. Based on the upgraded Politecnico di Milano (POLIMI) kinetic mechanism, the relative reactivity of n-butane and the different oxygenated fuels...

An experimental and theoretical kinetic study of the reaction of OH radicals with tetrahydrofuran

Tetrahydrofuran (C4H8O, THF) and its alkylated derivatives of the cyclic ether family are considered to be promising future biofuels. They appear as important intermediates during the low-temperature oxidation of conventional hydrocarbon fuels and of heavy biofuels such as long-chain fatty acid methyl esters. The reaction of tetrahydrofuran with OH radicals was investigated in a shock tube, over a temperature range of 800–1340 K and at pressures near 1.5 bar. Hydroxyl radicals were generated by the rapid thermal decomposition of tert-butyl hydroperoxide, and a UV laser absorption technique was used to monitor the mole fraction of OH radicals. High-level CCSD(T)/cc-pV(D,T)Z//MP2/aug-cc-pVDZ quantum chemical calculations were performed to explore the chemistry of the THF+OH reaction system. Our calculations reveal that the THF+OH (R1) reaction proceeds via either direct or indirect H-abstraction from various sites, leading to the formation of tetrahydrofuran-2-yl (THF-R2) or tetrahydrofuran-3-yl (THF-R3) radicals and water. Theoretical kinetic analysis revealed that both channels are important under conditions relevant to combustion. To our knowledge, this is the first direct experimental and theoretical kinetic study of the reaction of tetrahydrofuran with OH radicals at high temperatures. The following theoretical rate expressions (in units of cm3mol−1s−1) are recommended for combustion modeling in the temperature range 800–1350 K:k1(T)=4.11×104(TK)2.69exp(1316.8KT)(THF+OH→Products)k2(T)=6.93×1011(TK)0.41exp(−106.8KT)(THF+OH→THF-R2+H2O)k3(T)=4.12×103(TK)3.02exp(456.9KT)(THF+OH→THF-R3+H2O)

Kinetics of Non-Catalytic Esterification of Free Fatty Acids Present in Jatropha Oil.

Non-catalytic esterfication of free fatty acids (FFA) present in vegetable oils is an alternative pretreatment process for the biodiesel production. Biodiesel, consists of long-chain fatty acid methyl esters (FAME) and is obtained from renewable sources such as vegetable oils or animal fat. This study presents kinetics of thermal esterification of free fatty acids present in jatropha oil with methanol. The effect of process parameters like reaction time (1-5 h), temperature (170-190°C) and oil to methanol ratio (1:3-1:5) at constant pressure was investigated. The optimal conditions were found to be oil to methanol ratio of 1:4, 190°C, at 27.1 bar and 5 h which gave a maximum conversion of 95.1%. A second order kinetic model for both forward and backward reactions was proposed to study the reaction system. A good agreement was observed between the experimental data and the model values. The activation energy for forward reaction and the heat of reaction were found to be 36.364 Kcal/mol and 1.74 Kcal/mol respectively.

Catalytic hydroprocessing of fatty acid methyl esters to renewable alkane fuels over Ni/HZSM-5 catalyst

A series of Ni/HZSM-5 catalysts with different Ni loading and Si/Al ratios were prepared by incipient wetness impregnation. The physicochemical properties of the catalysts were investigated by X-ray diffraction (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM), N2-adsorption, temperature-programmed desorption of ammonia (NH3-TPD), and thermogravimetric (TG) analysis. Moreover, their catalytic performance was investigated for the hydroprocessing of long-chain unsaturated fatty acid methyl esters (FAMEs) to renewable alkane fuels using a high-pressure fixed bed reactor system with a facility for online analysis. The different Ni loading and Si/Al ratios of the catalysts, as well as the influence of reaction conditions such as temperature, pressure, H2/oil molar ratio, and liquid hourly space velocity (LHSV), were studied in detail. The NiO aggregates dispersed on the surface of the support clearly increased the acidity after H2 reduction, thus significantly affecting the catalytic performance. Temperature and pressure played crucial roles in the conversion of FAMEs and selectivity for gasoline or jet or diesel alkane. Hydroprocessing over 10wt% Ni/HZSM-5 (Si/Al=25) at 280°C, a H2 pressure of 0.8MPa, an LHSV of 4h−1, and with a highly purified H2/oil molar ratio of 15 led to a high selectivity of 88.2% for C5–C18 liquid alkanes, which includes 8% of gasoline alkane, 32.5% of jet alkane and 47.7% of diesel alkane, along with appropriate isomerization selectivity of 27.0%, while the conversion of FAME reached 85.1%. To demonstrate the potential of this catalyst for practical applications, its stability in the hydroprocessing of FAMEs was also investigated. The conversion of FAMEs decreased to 30.1% over 10wt% Ni/HZSM-5 (Si/Al=25) after operation for 80h. Catalyst deactivation was predominantly caused by the deposition of carbon which causes blockage of the pores for FAMEs as evidence from TG analysis.

Evaluation and Predictive Model Development of Oxidative Stability of Biodiesel on Storage

Biodiesel comprises mono-alkyl esters of long-chain fatty acid methyl esters (FAME) derived from a renewable lipid feedstock. A major technical issue with the use of biodiesel is that it is more prone to oxidation during storage, when compared to petroleum fuel, due to the high content of polyunsaturated methyl esters that are easily oxidizable to compounds such as acids, aldehydes, and alcohols. Biodiesel (Jatropha and Pongamia) and antioxidants (Turmeric and butylated hydroxytoulene) were used for this study. We found that the acid value and viscosity for pongamia biodiesel increased significantly by 41.17% and 44.0% and that for jatropha biodiesel increased by 31.5% and 37.0%, respectively, after being stored for 3 months. The impact of antioxidants on the storage stability of biodiesel was examined according to the ASTM D4625 12-week procedure, and best results were found at a concentration level of 2500 ppm. The specific objective of this investigation is to develop models to determine the viscosity of biodiesel at any time “t” during long-term storage based on these experimental trials for upto 12 weeks. In addition, the models were used to predict the level of antioxidants that are to be added to biodiesel in order to minimize the effects of oxidative degradation during storage. The developed model recorded an adjusted R2 of 0.86 and a modeling efficiency of 0.88.

Mixed Trophic State Production Process for Microalgal Biomass with High Lipid Content for Generating Biodiesel and Biogas

Economically feasible and sustainable energy production from microalgae requires optimization of algal growth, maximization of lipid content, and enhancement of biomass conversion into energy. An innovative, mixed trophic state process with high productivity was implemented to generate microalgae with high lipid content for generating biodiesel and biogas. Auxenochlorella protothecoides, a unicellular green alga, was grown phototrophically to 0.28 dry weight per L (gdw/L) then concentrated to 36 gdw/L for use as an inoculum for a subsequent heterotrophic cultivation to a final density of nearly 120 gdw/L. Simultaneous nitrogen deprivation and glucose supplementation during the heterotrophic stage increased the total lipid content from 16 to 57 % while the triacylglycerol (TAG) fraction of total lipids advanced from 2 to 79 %. Productivity peaked at 4.9 g of biomass/L-h and 1.7 g TAGs/L-h. The extracted lipids, including high levels of oleic, linoleic, and palmitic acids, were converted into biodiesel with a predicted cetane number of 56.4 and low concentrations of long-chain saturated and polyunsaturated fatty acid methyl esters. Both intact microalgal biomass and lipid-extracted algal residues (LEA) were good substrates for anaerobic digestion (AD) with methane yields of 0.6 and 0.4 L/g volatile solids (VS), respectively. These yields represented nearly 80 % of theoretical methane potential. LEA, with a favorable carbon to nitrogen ratio (C:N) of approximately 19:1, is an appropriate substrate for anaerobic microorganisms, most likely because it contains essential nutrients required for microbial digestion. The biochemical composition of the biomass, especially its lipid content, is the major contributor for energy output. As a result, coupling biodiesel production with AD of LEA to generate methane can increase the overall process’ energy output up to 40 %.

Study of the Effect of Storage Time on the Oxidation and Thermal Stability of Various Biodiesels and Their Blends

Biodiesel can be described as a safe alternative fuel, which can replace petroleum diesel in the future. It consists of long-chain fatty acid methyl esters (FAME). Biodiesel has high lubricity and is a clean burning fuel. It also produces less air pollution, is renewable biodegradable, and is safer for the environment. Since biodiesel is produced from vegetable oil, animal fats, used cooking oil, and so forth, which may contain more or less unsaturated fatty acids that are prone to oxidation accelerated by exposure to air during storage and at high temperature, it may yield polymerized compounds. The oxidation and thermal stability of the fuel changes with storage time due to the formation of oxidation. Therefore, the aim of this study to evaluate the stabilities of biodiesel according to measured fuel properties, such as density, viscosity, flash point, total acid number (TAN), and total base number (TBN), by using various methodologies. In addition, oxidation stability of the samples was measured by the induction period using a Rancimat instrument. In this experiment, palm oil methyl ester (PME), palm biodiesel blend (40% PME and 60% diesel fuel), jatropha methyl ester (JME), jatropha biodiesel blend (40% JME and 60% diesel fuel), coconut oil methyl ester (COME), and conventional diesel fuel were used. Experiments were carried out at intervals over a 12-week test period. The experimental results for JME and PME showed similar performance in terms of flash point. All samples met the standard specification of the American Society for Testing and Materials (ASTM) D6751. (3 h) regarding the induction period, except for JME and its biodiesel blend, which did not meet the EN 14214 (6 h) standard specification. Among the fuel samples giving the worst results for TBN value due to oxidation, overall, among the biodiesels, PME and COME were found to give better results with respect to oxidation and storage stabilities.

Investigations to check applicability of 1st generation biocomponents to fuels for turbine aircraft engines

The study outlines results from the studies on applicability of 1st generation biocomponent, namely long-chain fatty acid methyl esters (FAME) of vegetable origin, as an ingredient to fuels used to supply turbine aircraft engines. The presented analysis refers to both physical and chemical properties of developed fuel mixtures of Jet A-1 pure kerosene fuel with various amounts of the FAME additive and is based on own results obtained from tests of turbine engines on workbenches. The experiment results indicate that there are virtually no chances to apply the 1st generation biocomponents of the FAME type to aircrafts, however it is possible to seek for application opportunities of such biocomponents to other turbine engines, different from the aviation engineering.

Physicochemical characterization and formation of glyphosate-laden nano-emulsion for herbicide formulation

A nano-emulsion system was developed for pesticide formulation. Pseudoternary phase diagrams were constructed consisting emulsion system of long-chain fatty acid methyl esters (LFAMEs)/mixed surfactant/water and a quarternary component, glyphosate isopropylamine (IPA) as a herbicide active. Isotropic (L) regions were formed in the phase diagrams using mixed surfactant long-chain alkylpolyglucosides (LAPG) and ethoxylated 3-(3-hydroxypropyl)-heptamethyltrisiloxane (organosilicone) at the ratios of 9:1, 8:2 and 7:3. Pre-formulation concentrates were chosen from the L regions with less than 20% (w/w) of inerts (LFAMEs+mixed surfactant) and were characterized with regard to particle size, particle aging rate and thermostability study. A pre-formulation concentrate with the lowest aging rate and stable at high temperature (54°C) was selected for the mechanisms study of the pre-formulation concentrate in conjunction with the development of nano-emulsion formulation. The transmission electron microscopy (TEM) result showed that the pre-formulation concentrate appeared as a polymerized multi-connected network. Upon water dilution of the pre-formulation concentrate with gentle stirring (low-energy emulsification method), well-dispersed nanoparticles were formed with no needle structure being observed. The nano-emulsion particles were incorporated well with the glyphosate IPA thus inferring that this nano-emulsion system could ameliorate the bioactivity and bioavailability of the herbicide.

High-level expression of extracellular lipase Lip2 from Yarrowia lipolytica in Pichia pastoris and its purification and characterization

The extracellular lipase gene from Yarrowia lipolytica (YlLip2) was cloned into the pPICZαA and integrated into the genome of the methylotrophic yeast Pichia pastoris X-33. The lipase was successfully expressed and secreted with an apparent molecular weight of 39kDa using Saccharomyces cerevisiae secretion signal peptide (α-factor) under the control of the methanol inducible promoter of the alcohol oxidase 1 gene (AOX1). The lipase activity of 12,500,000U/l (2.10g total protein and 0.63g lipase per liter) was obtained in a fed-batch cultivation, where methanol feeding was linked to the dissolved oxygen content after initial glycerol culture. After fermentation, the supernatant was concentrated by ultrafiltration with a 10kDa cut off membrane and purified with ion exchange chromatography using Q Sepharose FF. Deglycosylation showed that the recombinant lipase is a glycoprotein which contains the same content of sugar (about 12%) as the native lipase from Y. lipolytica. The optimum temperature and pH of the recombinant lipase was 40°C and 8.0, respectively. The lipase showed high activity toward long-chain fatty acid methyl esters (C12–C16).

Determination of the carbon deficiency in the flame ionization detector response of long-chain fatty acid methyl esters and dicarboxylic acid dimethyl esters

Carbon deficiencies (CDs) of long-chain fatty acid methyl esters (FAMEs) and dicarboxylic acid dimethyl esters (DDMEs), which lead to decreased response in a flame ionization detection (FID) system, were determined by using full responding hydrocarbons (heptadecane, eicosane and alpha-cholestane) as references. For saturated FAMEs ranging from C 12 to C 22 and for DDMEs ranging from C 4 to C 10, CDs between 1.3 ± 0.12 and 1.7 ± 0.36 per ester group were recorded. All values were significantly ( P < 0.05) greater than 1. Generally, response factors for gas-chromatographic analysis using FID have been calculated on the theory that the CD of FAMEs is 1 per ester group. However, this theory could not be confirmed experimentally for short-chain FAMEs of less than 8 carbons as CDs of around 1.5 were reported for C 4 and C 6 FAMEs. The study presented here contributes an approach to this problem by confirming the validity of response factors calculated from a CD of 1.5 per ester group as well as for long-chain FAMEs and DDMEs.