The application of biotechnological methods for the synthesis of biodiesel

Synthesis of biodiesel is performed mainly by chemical catalysis, but can also be performed by enzymatic or microbial methods, and these might play an important role in future substitution of petroleum‐based diesel. To discover sustainable, economically attractive biotechnological processes for biodiesel synthesis, close cooperation between different disciplines is needed. Currently, lipases are the enzymes of choice for the synthesis of fatty acid esters (FAE) from fats and oils, yielding biodiesel with the methyl esters (FAME) as the most important product. More recently, the direct production of FAE using engineered whole cell microorganism has also been described (MicroDiesel). Current enzymatic processes are still hampered by the high costs of the biocatalyst, but significant progress has recently been made leading to the first industrial enzymatic biodiesel production. Enzymatic biodiesel production is mostly attractive because of the starting materials (waste frying oils, oils with high water content, etc.), for which conventional chemical interesterification can hardly be applied.     

Oxidative stability of structured lipids produced from borage (<Emphasis Type="Italic">Borago officinalis</Emphasis> L.) and evening primrose (<Emphasis Type="Italic">Oenothera biennis</Emphasis> L.) oils with docosahexaenoic acid

This study utilized γ-linolenic acid (18∶3n−6; GLA)-rich borage oil (BO) and evening primrose oil (EPO) for the synthesis of structured lipids (SL) and compared the oxidative stability of the products with those of unmodified BO and EPO as controls. Immobilized Novozym 435 lipase from Candida antarctica was used as the biocatalyst for SL production. BO or EPO eas enzymatically modified with docosahexaenoic acid (22∶6n−3; DHA), as the acyl donor, to produce SI. The SI were characterized and their oxidative stabilities evaluated. Among the oils examined, SL gave rise to higher quantities (P≤0.05) of conjugated dienes, TBARS, and headspace volatiles as compared to their unmodified counterparts. Results indicated that modified oils were less stable than their unmodified counterparts. The double bond index (DBI) and methylene bridge index (MBI) of oils decreased (P<0.05) during oxidation in the more unsaturated oils. An attempt was made to correlate various parameters of oxidation with DBI and MBI of oils; correlation coefficients (−r) were within the range of 0.574–0.973. This suggests that indicators such as DBI and MBI can reflect oxidative stability of oils.     

L(+)‐Lactic acid production using poly(vinyl alcohol)‐cryogel‐entrapped Rhizopus oryzae fungal cells

A new immobilized biocatalyst based on Rhizopus oryzae fungal cells entrapped in poly(vinyl alcohol)‐cryogel was evaluated in both the batch and semi‐batch processes of L (+)‐lactic acid (LA) production, when glucose, acid hydrolysates of starch or gelatinized potato starch were used as the main substrates. Under the batch conditions, the immobilized biocatalyst developed produced LA with yields of 94% and 78% from glucose and acid starch hydrolysates, respectively. Semi‐batch conditions enabled product yields of 52% and 45% to be obtained with the corresponding substrates. The highest process productivity (up to 173 g L^?1) was reached under semi‐batch conditions. Potato starch (5–70 g L^?1) was also transformed into lactic acid by immobilized R. oryzae. It was shown that long‐term operation of the immobilized biocatalyst (for at least 480 h) produced a low decrease in metabolic activity. Copyright © 2006 Society of Chemical Industry     

Canola Quality Indian Mustard Oil (<Emphasis Type="Italic">Brassica juncea</Emphasis>) is More Stable to Oxidation than Conventional Canola Oil (<Emphasis Type="Italic">Brassica napus</Emphasis>)

Canola-quality Indian mustard (Brassica juncea) is being developed as a complimentary oilseed crop to canola (Brassica napus) for cultivation in hot and low-rainfall areas, where canola does not perform well. In Australia, several B. juncea breeding lines have been developed for commercial cultivation and for eventual processing as canola oil. However, there still are significant species-based differences in the fatty acid composition with B. juncea containing lower levels of linoleic acid and higher levels of oleic and linolenic acids compared with B. napus. This has raised concern about possible oxidative stability differences between the oils. Oils (unrefined) extracted from different breeding lines of each species were subjected to accelerated autoxidation, and development of oxidative rancidity was assessed by four separate techniques: depletion of polyunsaturated fatty acids, depletion of tocopherol, development of primary oxidation products, and development of secondary oxidation products (propanal and hexanal). All the tests showed that the newly developed B. juncea oils are more stable to autoxidation than conventional canola (B. napus) oil, despite containing marginally higher linolenic levels. Oxidative stability does not appear to be a barrier to using oils from these emerging lines of B. juncea for partial or full replacement of conventional canola oil.     

Efficient cell surface display of organophosphorous hydrolase using N-terminal domain of ice nucleation protein in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Recombinant Escherichia coli systems expressing organophosphorous hydrolase (OPH) have been used for detoxifying toxic organophosphate compounds. However, a whole cell biocatalyst system has an intrinsic problem due to substrate diffusion limitation by its cell membrane. As a strategy for reducing this diffusion barrier limitation to enhance whole cell biocatalytic activity, we engineered E. coli cells to target OPH on cell surface using ice nucleation protein (InaK) as a surface targeting motif, especially N-terminal domain of InaK (InaK-N). The whole cell OPH activities of the cells expressing InaK/OPH fusion constructs were higher (∼2.5-fold for InaK-N and ∼5.7-fold for combined N-and C-terminal domain of InaK (InaK-NC)) than that of the cells expressing cytosolic OPH. Interestingly, the membrane targeting efficiency of the cells expressing InaK-N/OPH fusion proteins was ∼2.2-fold higher compared to the cells expressing InaK-NC/OPH even though both whole cell and total cell lysate OPH activities were lower. Therefore, we found that the small size N-terminal domain of InaK is more efficient for targeting OPH on the cell surface, and the surface display of OPH using N-terminal InaK domain can reduce the mass-transfer problem in whole cell bioconversion system. This work was presented at 13 ^th YABEC symposium held at Seoul, Korea, October 20–22, 2007     

Quantitative determination of diesel oil in contaminated edible oils using high-performance liquid chromatography

A simple and reliable high-performance liquid chromatography method for the analysis of diesel oil in contaminated edible oils is described. Analysis performed using a diol column with a mobile phase of heptane and isopropanol (94∶6, vol/vol). Although baseline separation between diesel and other background fluorescent components was not achieved, quantitation was still possible using baseline integration. The method is linear over the range of 5–1000 μg/g with a correlation coefficient (r ²) of 0.9984. Average recoveries from spiked edible oils were 94.4–101.3%, with a limit of quantitation (LOQ) of 5 μg/g for sunflower oil, palm olein, and groundnut oil. Corn oil has a higher content of ester components, thus, LOQ was slightly worse (40 μg/g). The applicability of the method was confirmed by gas chromatography-mass spectroscopic detection to show the presence of diesel hydrocarbons in the suspected contaminated crude palm oil. This procedure provides a simple and sensitive method for determining diesel oil concentration in contaminated edible oils without prior sample cleanup or extraction.     

Immobilization of Saccharomyces cerevisiae in Gelatin Cross-Linked with Chromium Ions for Conversion of Sucrose by Intracellular Invertase

A novel immobilized biocatalyst with invertase (β-D-fructofuranosidase, E.C.3.2.1.26) activity was prepared by immobilizing Saccharomyces cerevisiae cells into gelatin by using chromium salts. The effect of chromium salts on cell growth of free and immobilized cells was investigated, and optimum concentrations for chromium acetate and chromium sulfate were found to be 0.016 M and 0.008 M, respectively. Since a medium feeding strategy was applied, higher enzymatic activities were obtained with developed biocatalyst compared to free S. cerevisiae cells. Immobilized samples could be used 10 times in a 30-day period with negligible activity loss. After storing it at 25°C for 28 days, our biocatalyst was used 15 times with 2-day intervals with only a slight amount of activity decrease. As a result we managed to produce a very stable biocatalyst with high invertase activity using an inexpensive methodology.     

Sulfur specificity in the bench‐scale biological desulfurization of crude oil by Rhodococcus IGTS8

Biological removal of organic sulfur from petroleum feedstocks may offer an attractive alternative to conventional thermochemical treatment due to the mild operating conditions and greater reaction specificity afforded by the nature of biocatalysis. Previous investigations have either reported the desulfurization of model sulfur compounds in organic solvents or gross desulfurization of crude oil without data on which sulfur species were being removed. This study reports initial sulfur speciation data for thiophenic sulfur compounds present in crude oil which may be used as a guide both as to which species are treated by the biocatalyst investigated as well as to where biocatalyst development is needed to improve the extent of biological desulfurization when applied to whole crudes. Biodesulfurization of two different crude oils in the 22–31 ° API specific gravity range with total sulfur contents between 1 and 2% is demonstrated in 1‐dm³ batch stirred reactors using wild type Rhodococcus sp IGTS8. While analysis of the crudes before and after biodesulfurization did not reveal a decrease in total sulfur, GC–MS did reveal significant (43–99%) desulfurization of dibenzothiophenes (DBT) and substituted DBTs. Fractionation of the whole crude, followed by analysis using gas chromatography–sulfur chemiluminescence detection (GC–SCD) of the aromatic fraction of the Van Texas crude oil, demonstrated a reduction of sulfur in this fraction from 3.8% to 3.2%. This research indicates that IGTS8 may be capable of biodesulfurization of refined products such as gasoline and diesel whose predominant sulfur species are dibenzothiophenes. Further biocatalyst development would be needed for effective treatment of the spectrum of sulfur‐bearing compounds present in whole crudes.     

Asymmetric reduction of chloroacetophenones to produce chiral alcohols with microorganisms

Four strains of yeast with reduction activity of chloroacetophenones were screened, in which Saccharomyces cerevisiae B5 showed best reduction activity and stereoselectivity. High optical purity (R)-2′-chloro-1-phenylethanol can be obtained with Saccharomyces cerevisiae B5 as biocatalyst. The influence of several co-substrates on the enantiometric excess (ee%) and yield of (R)-2′-chloro-1-phenylethanol was evaluated. 5% (v/v) ethanol is optimal cosubstrate for (R)-2′-chloro-1-phenylethanol formation. The optimal bioconversion conditions of 2′-chloroacetophenone catalyzed by Saccharomyces cerevisiae B5 are as follows: pH 8.0, 25 °C and 24 h. The yield and the enantiometric excess of (R)-2′-chloro-1-phenylethanol can both reach more than 99% with 10.75 g/l Saccharomyces cerevisiae B5 (the cell dry weight) and 1 g/l 2′-chloroacetophenone used in the biotransformation.     

Increased generation of electricity in a microbial fuel cell using <Emphasis Type="Italic">Geobacter sulfurreducens</Emphasis>

The microbial fuel cell (MFC) has attracted research attention as a biotechnology capable of converting hydrocarbon into electricity production by using metal reducing bacteria as a biocatalyst. Electricity generation using a microbial fuel cell (MFC) was investigated with acetate as the fuel and Geobacter sulfurreducens as the biocatalyst on the anode electrode. Stable current production of 0.20–0.24 mA was obtained at 30–32 °C. The maximum power density of 418–470 mW/m², obtained at an external resistor of 1,000 Ω, was increased over 2-fold (from 418 to 866 mW/m²) as the Pt loading on the cathode electrode was increased from 0.5 to 3.0 mg Pt/cm². The optimal batch mode temperature was between 30 and 32 °C with a maximum power density of 418–470 mW/m². The optimal temperature and Pt loading for MFC were determined in this study. Our results demonstrate that the cathode reaction related through the Pt loading on the cathode electrode is a bottleneck for the MFC’s performance.     

<Emphasis Type="Italic">Trans</Emphasis> FA in sunflower oil at different steps of refining

The contents of total trans FA of sunflower oils at different stages of refining processes were determined by capillary GLC. The contents of 18∶1, 18∶2, and 18∶3 trans acids were 0.22±0.03, 2.31±0.23, and 0.03±0.01%, respectively, in physically refined sunflower oils, and 0.05±0.01, 0.69±0.26, and 0.02±0.01%, respectively, in chemically refined sunflower oils. The total trans FA contents drastically increased at the end of the physical refining process. The total trans FA contents of chemically refined sunflower oils were <1%. Because of the high temperature applied in the last stage of physical refining, the content of total trans FA was higher than in chemically refined sunflower oils. The last-stage conditions should be carefully evaluated to reduce the formation of trans FA during physical refining.     

Lactic acid production using free cells of bacteria and filamentous fungi and cells immobilized in polyvinyl alcohol cryogel: A comparative analysis of the characteristics of biocatalysts and processes

The results from studies and a comparative analysis of process characteristics are presented for lactic acid (LA) production from glucose, performed under batch conditions of the long-duration use of different biocatalyst samples comprising cells of Lactobacillus сasei bacteria and Rhizopus oryzae filamentous fungi immobilized in polyvinyl alcohol (PVA) cryogel or applied as concentrated suspensions. It is established that for LA production, the bacteria and fungi must be used in the form of PVA-cryogel-immobilized cells because their half-life in this form is considerably longer than that of concentrated cell suspensions. After 200 h of the batch use of the same immobilized cell samples, the amounts of accumulated LA were similar for both fungal (920 ± 5 g) and bacterial (895 ± 5 g) biocatalysts. The fungal biocatalyst, however, was characterized by a twice higher rate of substrate conversion to product (0.92 g LA per 1 g glucose) than the bacterial biocatalyst. The half-life of the immobilized fungal biocatalyst was 80 days (96 working cycles), ten times longer than that of the bacterial biocatalyst. A comparison of our data and the literature data demonstrated the promise of using fungal cells immobilized in PVA cryogel to produce LA: the process based on their use is superior to all known processes in its main indicators, i.e., the rate of LA conversion to glucose and the maximum accumulated concentration of the product.     

Removal of ethene to very low concentrations by immobilised mycobacterium E3

Removal of ethene to concentrations below 1 vpm from the air in storage facilities is necessary to prevent the deterioration of stored fruits, vegetables and flowers. Ethene-utilising Mycobacterium E3 organisms are able to oxidise ethene to these low concentrations. Ethene was oxidised at the same rate by organisms immobilised on lava or perlite as it was by the suspension of cells. The residence times in a gas/solid bioreactor necessary to convert 66% of the ethene with an initial concentration of 3.2 vpm, and with cell loads on lava of 0.72 or 0.36 mg protein g^?1 support, were 15 s and 33 s respectively. Mycobacterium E3 organisms immobilised on lava lost half of their activity in 250 h while organisms immobilised on perlite lost half of their activity in 60 h. Although the operational stability of the biocatalyst should be improved and the biocatalyst is only suitable at temperatures above 10°C, the application of ethene-utilising bacteria in a gas/solid bioreactor appears attractive.     

Lipid body formation by <Emphasis Type="Italic">Thraustochytrium aureum</Emphasis> (ATCC 34304) in response to cell age

The heterotrophic marine protist, Thraustochytrium aureum produces substantial amounts of polyunsaturated fatty acids (PUFAs). In the present investigation, changes in the lipid and fatty acid profiles of T. aureum were studied according to the culture age. T. aureum was grown in artificial sea water medium for 10 days at 25 °C in shake culture condition. One to 10 day old cell samples were analyzed for cell biomass production, total lipid content, fatty acid profile and lipid body formation. In all the samples tested, total lipid production was found to be directly proportional to the dry cell weight of T. aureum. In the early phase of cell growth, cell biomass production, lipid content and glucose consumption were found to be higher. Thin layer chromatographic analysis (TLC) of lipids showed the presence of triacylglycerol (TAG; 169 mg/g, 90%), phospholipids (PL; 83 mg/g, 66%) and sterol (ST; 6 mg/g, 5%), which were recorded at maximum levels in the early growth phase of the cells. The composition of PUFAs and saturated fatty acids (SFAs) of the cell biomass and lipid class components (TAG and PL) was identified by gas chromatographic analysis (GC). In the early phase of cell growth, production of PUFAs in the total fatty acids was found to have attained maximum levels (61.3%) in which docosahexaenoic acid alone showed higher content of occurrence (99.0 mg/g in total lipid; 65.2 mg/g in TAG and 41.0 mg/g in PL). In the middle phase of cell growth, palmitic acid production was found to be higher (36.7 mg/g in total lipid; 31.3 mg/g in TAG and 12.6 mg/g in PL). Transmission electron microscopic studies of the cells showed the presence of a membrane around the lipid bodies in the early phase of cell growth. TAG and PL were actively involved in the formation of lipid bodies in the cells of T. aureum. Large-sized lipid bodies accumulated in 3 day old cells which were then fragmented into smaller bodies in the late growth phase.     

Processing efficiency of immobilized non-growing bacteria: Biocatalytic modeling and experimental analysis

Modeling methods used to optimize the biocatalytic efficiency of freely suspended cells have been applied to non-growing microbial cells entrapped within a macro-porous carrier. The catalytic rate, which is dependent on cell concentration inside the biocatalyst beads, coincided with catalytic parameters for freely suspended cells. Immobilized non-growing cell systems could be optimized utilizing the characteristics of freely suspended cells without requiring extensive experimentation to define catalytic behaviour inside the biocatalyst. A dynamic diffusion–reaction model was developed and validated using experimental data for thiodiglycol degradation by Alcaligenes xylosoxidans subsp. xylosoxidans immobilized within macro-porous poly(vinyl alcohol) cryogel in a completely mixed batch bioreactor.     

Performance and mechanism of a hydrophobic–oleophilic kapok filter for oil/water separation

A natural vegetable fiber, kapok, was evaluated for its feasibility as filter material for separating immiscible liquids such as oil/water mixtures. Two types of oils, namely diesel and hydraulic, were investigated. Various simulated oily waters were prepared by mixing tap water with one of these oils to produce binary oil/water mixtures of varying oil percentages, such as 5%, 10% and 15% by weight. The effect of filter packing density on the dynamics of oil/water separation during the filtration process and the filtrate quality were investigated. Due to the hydrophobic–oleophilic surface characteristic of the kapok fiber, water could be filtered through the filter while oils were retained, causing separation of oil from water. The filtration efficiency for removal of diesel and hydraulic oil from their respective oily waters reached 100% and more than 99.4%, respectively, which showed an excellent performance of kapok in removing oils from oily waters. The hydraulic conductivity of the kapok filter was found to decrease with the advance of the oil front. The filtration rate of both types of oily waters decreased with increasing filter packing densities and influent oil contents. The kapok filter performed equally well in terms of total filtrate collected before breakthrough for the two different oils, albeit the filtrate rate could be viscosity dependent. A packing density of 0.07 g/cm³ was the optimum one for diesel and hydraulic oil removal. At breakthrough, the oil/water/air three-phase partition within the filter bed is a function of influent oil concentration, oil type, and packing density of the filter.     

Co‐immobilized Whole Cells with ω‐Transaminase and Ketoreductase Activities for Continuous‐Flow Cascade Reactions

An improved sol–gel process involving the use of hollow silica microspheres as a supporting additive was applied for the co‐immobilization of whole cells of Escherichia coli with Chromobacterium violaceum ω‐transaminase activity and Lodderomyces elongisporus with ketoreductase activity. The co‐immobilized cells with two different biocatalytic activities could perform a cascade of reactions to convert racemic 4‐phenylbutan‐2‐amine or heptan‐2‐amine into a nearly equimolar mixture of the corresponding enantiomerically pure R amine and S alcohol even in continuous‐flow mode. The novel co‐immobilized whole‐cell system proved to be an easy‐to‐store and durable biocatalyst.     

A Comparative Study of the Properties of Selected Melon Seed Oils as Potential Candidates for Development into Commercial Edible Vegetable Oils

A comprehensive compositional and characterization study was carried out on five seed oils from varieties of the melons Citrullus lanatus and C. colocynth in order to evaluate their suitability for large-scale exploitation as edible vegetable oils. The oils were extracted by Soxhlet with a 3:1 mixture of n-hexane/2-propanol with yields that ranged from 24.8 to 30.0% (wt/wt). The refractive indices and relative densities of the oils fell within the narrow ranges of 1.465–1.469 and 0.874–0.954 g/cm³, respectively. Saponification values ranged between 182.1 and 193.8 mg KOH/g, whilst iodine values (IV) ranged from 95.8 to 124.0 (Wijs). The ranges of the values for free fatty acid (AV), 1.2–4.0 mg KOH/g, peroxide (PV), 1.1–10.9 meq/kg and p-anisidine (p-AV), 0.2–9.0, indicated that secondary oxidation products were barely present. GC analysis gave total unsaturation contents of 67.93–82.36%, with linoleic acid (18:2) being the dominant fatty acid (55.21–66.85%). The GC results agreed closely with those from proton NMR analysis of the fatty acid classes. The physicochemical and compositional properties determined in this study show that the qualities of the test Cucurbitacea seed oils are highly comparable to those of soybean, sunflower and groundnut seed oils. Therefore, the test melon seed oils could be developed into commercial products to serve as alternate vegetable oils in Southern and West Africa, the regions where these melons grow.     

Biocatalytic production of extracellular exopolysaccharide dextran synthesized by cells of <Emphasis Type="Italic">Leuconostoc mesenteroides</Emphasis>

Results are presented from studies and a comparative analysis of the production of the commercially important product dextran from sucrose using fed-batch cultivated cells of the Leuconostoc mesenteroides subsp. dextranicum B-5481 bacterium either immobilized in a polyvinyl alcohol (PVA) cryogel or in the form of a suspension. It is shown that under identical process conditions, the concentration of dextran is 1.2 times higher when using immobilized cells instead of free cells. The high productivity of dextran formation (4.2 g/(L h)) under the conditions of fed-batch cultivation of the immobilized cells and the ability of these cells to function without losing their metabolic activity for at least five operating cycles are demonstrated. The productivity of the developed biocatalyst is 5 times higher than that of Weissella confusa cells immobilized in a calcium alginate gel and 34 times higher than that of Leuconostoc mesenteroides KIBGE HA1 cells immobilized in a polyacrylamide gel. The molecular weight of the dextran samples produced by the immobilized L. mesenteroides B-5481 cells is half that of the polymer produced by the free cells, expanding the range of possible applications of the polysaccharide with no additional hydrolysis.     

Photoautotrophic cultivation of the green alga Chlamydomonas reinhardtii as a method for carbon dioxide fixation and α-linolenic acid production

Focusing on CO₂ fixation and α-linolenic acid (ALA) production, photoautotrophic cultivation of the green alga Chlamydomonas reinhardtii was investigated by using a culture medium of pH 6.8 under a 5 vol% CO₂-enriched atmosphere. The optimum cultivation temperature and light intensity for growth were 25°C and 12 klux, respectively. The cellular ALA content nearly doubled to 11.9 mg/g of dry cells when the concentration of culture medium was doubled. Simulation of chemostat cultivation showed that the rate of CO₂ fixation and ALA productivity per unit volume of culture medium could reach 1.01 kg CO₂/(m³ · d) and 7.46 g ALA/(m³ · d), respectively, at a cell concentration of 0.57 kg cells/m³.