Multi-parameter flow cytometry as a tool to monitor heterotrophic microalgal batch fermentations for oil production towards biodiesel

Multi-parameter flow cytometry was used to monitor cell intrinsic light scatter, viability, and lipid content of Chlorella protothecoides cells grown in shake flasks. Changes in the right angle light scatter (RALS) and forward angle light scatter (FALS) were detected during the microalgal growth, which were attributed to the different microalgal cell cycle stages. The proportion of cells not stained with PI (cells with intact cytoplasmic membrane) was high (> 90%) during the microalgal growth, even in the latter stationary phase, suggesting that the microalgal cells built-up storage materials which allowed them to survive under nutrient starvation, maintaining their cytoplasmic membranes intact. A high correlation between the Nile Red fluorescence intensity measured by flow cytometry and total lipid content assayed by the traditional lipid extraction method was found for this microalga, making this method a suitable and quick technique for the screening of microalgal strains for lipid production, optimization of biofuel production bioprocesses, and scale-up studies. The highest oil content (∼28% w/w dry cell weight, estimated by flow cytometry) was observed in the latter stationary phase. In addition, C. protothecoides oil also depicted the adequate fatty acid methyl ester composition for biodiesel purposes at this growth phase, suggesting that the microalgal oil produced during the latter stationary phase could be an adequate substitute for diesel fuel. Medium growth optimization for enhancement of microalgal oil production is now in progress, using the multi-parameter approach.     

Characterization of fatty acid composition in healthy and bleached corals from Okinawa, Japan

Under bleaching conditions, corals lose their symbiotic zooxanthellae, and thus, the ability to synthesize fatty acids (FAs) from photosynthetically derived carbon. This study investigated the lipid content and FA composition in healthy and bleached corals from the Odo reef flat in Okinawa, southern Japan, following a bleaching event. It was hypothesized that the FA composition and abundance would change as algae are lost or die, and possibly microbial abundance would increase in corals as a consequence of bleaching. The lipid content and FA composition of three healthy coral species (Pavona frondifera, Acropora pulchra, and Goniastrea aspera) and of partially bleached and completely bleached colonies of P. frondifera were examined. The FA composition did not differ among healthy corals, but differed significantly among healthy, partially bleached, and completely bleached specimens of P. frondifera. Completely bleached corals contained significantly lower lipid and total FA content, as well as lower relative amounts of polyunsaturated FAs and higher relative amounts of saturated FAs, than healthy and partially bleached corals. Furthermore, there was a significantly higher relative concentration of monounsaturated FAs and odd-numbered branched FAs in completely bleached corals, indicating an increase in bacterial colonization in the bleached corals.     

Variation in lipid composition of some deep-sea fish (Teleostei: Oreosomatidae and Trachichthyidae)

The lipid, fatty acid and fatty alcohol compositions were determined for muscle samples from six species of deep-sea oreo collected from Australian waters; namely Neocyttus rhomboidalis, Neocyttus sp., Allocyttus verrucosus, Allocyttus niger, Pseudocyttus maculatus, and Oreosoma atlanticum. Neocyttus helgae, landed in North Atlantic waters, was also analysed. Similar analyses were also carried out on the muscle and swim bladder of the orange roughy Hoplostethus atlanticus from both Australian and North Atlantic waters. Orange roughy is currently a major commercial species in southern Australia and is a new-fishery in the North Atlantic; there are four species of oreo of increasing commercial significance in Australia due to orange roughy quota reductions. It is therefore necessary to determine if the oreo fishing industry is capable of supplementing the current orange roughy requirements with respect to muscle and oil demand. In the oreos, the mean lipid content ranged from 0.5 to 3% of wet weight, with a mixed lipid composition including wax ester, triacylglycerol, sterol and polar lipid. The ratio of the monounsaturated fatty alcohols 22:1 to 20:1 allowed samples from the two geographical regions to be distinguished. Total wax ester in muscle from North Atlantic male orange roughy was much higher than in Australian fish (27 vs. 8.5% wet weight, respectively); females from both locations contained similar amounts of wax ester (4.5 vs. 3.3%, respectively). Selected swim bladders from North Atlantic and Australian orange roughy show similar wax ester content (90 vs. 82%, respectively). The ratio of 22:1 to 20:1 fatty alcohols in orange roughy from the two regions was 0.5 (Australian) and 1.4 (North Atlantic). Indeed differences exist between oreos from the two locations, but not between orange roughy and this requires further investigation. With respect to the nutritional value, the oreos are more attractive than the orange roughy however lipid levels remain much lower compared with other popular species.     

Fatty acid composition of lipids from mushrooms belonging to the family Boletaceae

The non-polar lipid content and fatty acid (FA) composition of 11 mushroom species of the family Boletaceae were determined. The non-polar lipid content ranged from 2.0 (Leccinum aurantiacum and Boletus erythropus) to 5.4 % (w/w) d.w. (Suillus grevillei) with an average value of 2.9 %. More than 25 different FAs were found in the mushroom lipids. Unsaturated FAs, mainly linoleic and oleic acids, accounted for about 83 % of the total FAs, while palmitic acid was the main saturated FA. Some FAs are identified for the first time in Boletaceae and in higher Basidiomycetes (cis-11,12-methyleneoctadecanoic acid, 7-cis,10-cis hexadecadienoic) or in fungi (cis-11,12-methyleneoctadecanoic acid). There were significant differences (P < 0.05) in the contents of specific FAs between mushroom species.     

Distribution of alkanes and fatty acids in theCondalia montana (Rhamnaceae) species complex

The alkane and fatty acid composition of the lipid extracts from leaves of 14 populations ofCondalia: C. microphylla f.xanthocarpa, C. microphylla f.erythrocarpa, C. microphylla f.melanocarpa, C. montana, andC. buxifolia, were determined by gas chromatography. Seventeen alkanes and 14 fatty acids were investigated and a predominance of nonacosane, hentriacontane, palmitic and linoleic acids in all species was found.     

Lipid content and fatty acid composition in hypogeous organs ofHelleborus species (Ranunculaceae)

Root and rhizome of fourHelleborus species:H. viridis, H. odorus, H. niger, H. foetidus were examined throughout a two-year period in order to determine the variation of lipid content and fatty acid composition during the ontogenetic cycle. In the deciduous geophytesH. viridis andH. odorus subsp.laxus the lipid content reaches its maximum during the quiescent phase, whereas in the evergreen geophyteH. niger the lipid content is highest during the main growth period. In the chamaephyteH. foetidus lipid content is always very low and it does not show variation throughout the year. In all species the fatty acids detected in the neutral fraction are myristic, palmitic, stearic, oleic and linoleic acid. Palmitic and linoleic acid are always present in larger quantities. The pattern of the relative content of saturated/unsaturated fatty acids seems to be linked to the life form of these species.     

Evaluation of extraction methods for recovery of fatty acids from Botryococcus braunii LB 572 and Synechocystis sp. PCC 6803

Microalgae are a very diverse group of organisms that consist in both prokaryotic and eukaryotic forms. Some species of microalgae can be induced to overproduce particular fatty acids through simple manipulations of the physical and chemical properties of the culture medium. In this paper, the effect of different extraction techniques on the recovery of fatty acids from the freeze-dried biomass from two lipid-producing microalgal strains: Botryococcus braunii LB 572 (green algae) and Synechocystis sp. PCC 6803 (cyanobacteria) was examined. Five procedures were used: after conversion of the lipid material into the corresponding fatty acid methyl esters (FAMEs) via suitable derivatization reactions (extraction-transesterification) and direct transesterification of biomass to produce FAMEs (without the initial extraction step) that used differential types of catalysts and processing conditions. This study has shown that procedure 3, a one step practical procedure for lipid extraction and in situ methyl ester derivation could be used successfully for the determination of the fatty acid compositions of microalgae and cyanobacteria.     

Dr Han L. Golterman,a Limnologist: a tribute on the occasion of his 65th birthday and retirement

The green algae D. tertiolecta, the flagellate I. galbana and the diatom C. gracilis were grown in batch cultures. The organisms were analysed for lipid class composition at the logarithmic and stationary growth phases using the Chromarod-Iatroscan thin layer chromatography with flame ionization detection (TLC-FID) system.There were major differences in lipid class production among the organisms investigated, but few differences in lipid class distribution between log phase and stationary phase cultures of D. tertiolecta and I. galbana. C. gracilis displayed the general trend exhibited in diatom metabolism, which can be characterized by an increase in triacylglycerol synthesis in situations of stress.     

Lipid extracts from different algal species:1H and13C-NMR spectroscopic studies as a new tool to screen differences in the composition of fatty acids,sterols and carotenoids

One- and two-dimensional¹H- and¹³C-NMR spectra of lipid extracts fromUlva rigida, Gracilaria longa, Fucus virsoides andCodium tomentosum collected in the northern Adriatic Sea allowed screening of the content of fatty acid chains, carotenoids, free and acylated cholesterol and chlorophylls. The carotenoid-to-polyunsaturated fatty acid molar ratio was taken as a comparison parameter in samples ofUlva rigida collected in differentloci and seasons; the value was markedly higher in samples from the Lagoon of Venice than from marine coastal waters. The total cholesterol concentration was evaluated by¹H-NMR spectroscopy and similar values were found for all species. Two-dimensional heterocorrelated NMR spectroscopy was shown to give characteristic fingerprints of the lipid extracts from algal samples as regards the content in chlorophylls, unsaturated fatty acids and carotenoids.author for correspondence     

Therapeutic efficacy of Kalpaamruthaa on reactive oxygen/nitrogen species levels and antioxidative system in mammary carcinoma bearing rats

Reactive oxygen species play an important role in cancer and metastasis. Kalpaamruthaa is a modified Siddha preparation, which has been formulated in our laboratory. The preparation is an amalgamation of Semecarpus anacardium (SA), Emblica officinalis (EO) and honey, which gives an extra protectiveness to mammary carcinoma bearing animals (Sprague-Dawley stains were used for this study). The aim of our research is to determine the therapeutic efficiency of the drug with respect to lipid peroxidation and antioxidant status. The levels of lipid peroxides and antioxidant levels were measured in blood, and vital organs (liver, kidney and breast tissue) of control and experimental animals. In cancer condition, the LPO was increased and antioxidant levels were decreased. On drug (SA and KA) administration, decreased LPO and increased antioxidant levels were seen in control and experimental animals. This may be due to additive property of the drugs (SA, Emblica and honey), which possesses anticancer effect. The present study shows the good therapeutic efficacy of Kalpaamruthaa against mammary carcinoma.     

Antioxidant effect of the marine algae Chlorella vulgaris against naphthalene-induced oxidative stress in the albino rats

Alcoholic extract of the marine algae Chlorella vulgaris was examined for its free radical scavenging effect with reference to naphthalene-induced lipid peroxidation in serum, liver, and kidney of rats. Initially, upon naphthalene intoxication (435 mg/kg body weight, intraperitoneally), the lipid peroxidation activity increased significantly (P < 0.001), and in contrast, the enzymic antioxidants (superoxide dismutase, catalase, glutathione peroxidase) and non-enzymic antioxidants (glutathione, ascorbic acid, and α-tocopherol) levels decreased remarkably. When the naphthalene stressed rats were treated with Chlorella vulgaris extract (70 mg/kg body weight, orally), the lipid peroxidation activity reduced significantly (P < 0.001) and the activities of both the enzymic and non-enzymic antioxidants increased reaching near control values. The minimum concentration (70 mg/l) of the extract that exhibited maximum (85%) free radical scavenging activity was chosen for the experimental study. The present results suggest that Chlorella vulgaris extract exerts its chemo-preventive effect by modulating the antioxidants status and lipid peroxidation during naphthalene intoxication.     

Lipid and hydrocarbon production byBotryococcus spp. under nitrogen limitation and anaerobiosis

The production of lipids and hydrocarbons in batch cultures of the algaeBotryococcus braunii andB. protuberans has been studied with respect to nitrogen limitation under aerobic and anaerobic conditions. Nitrogen deficiency significantly decreased the dry weight, chlorophylla and protein contents but the amounts of carotenoids, carbohydrates and lipids increased in both the species. Nitrogen starvation gave a 1.6-fold increase in lipid content. Anaerobiosis under nitrogen deficient conditions gave greater lipid production than anaerobiosis in nitrogen supplemented medium. Under nitrogen deficiency, the hydrocarbon fraction increased and the polar lipids decreased. Anaerobiosis induced hydrocarbon synthesis more significantly than nitrogen deficiency but decreased other non-polar and polar lipids.     

Lipid Formation by Aqueous Fischer-Tropsch-Type Synthesis over a Temperature Range of 100 to 400 °C

The formation of lipid compounds during anaqueous Fischer-Tropsch-type reaction was studied withsolutions of oxalic acid as the carbon and hydrogensource. The reactions were conducted in stainlesssteel vessels by heating the oxalic acid solution atdiscrete temperatures from 100 to 400 °C, atintervals of 50 °C for two days each. Themaximum lipid yield, especially for oxygenatedcompounds, is in the window of 150–250 °C. At atemperature of 100 °C only a trace amount oflipids was detected. At temperatures above150 °C the lipid components ranged from C₁₂to >C₃₃ and included n-alkanols, n-alkanoic acids, n-alkyl formates, n-alkanals, n-alkanones, n-alkanes, andn-alkenes, all with essentially no carbon numberpreference. The n-alkanes increased inconcentration over the oxygenated compounds attemperatures of 200 °C and above, with a slightreduction in their carbon number ranges due tocracking. It was also noted that the n-alkanoicacids increased while n-alkanols decreased withincreasing temperature above 200 °C. Attemperatures above 300 °C synthesis competeswith cracking and reforming reactions. At 400 °Csignificant cracking was observed and polynucleararomatic hydrocarbons and their alkylated homologswere detected. The results of this work suggest thatthe formation of lipid compounds by aqueous FTTreactions proceeds by insertion of a CO group at theterminal end of a carboxylic acid functionality toform n-oxoalkanoic acids, followed by reductionto n-alkanoic acids, to n-alkanals, thento n-alkanols. The n-alkenes areintermediate homologs for n-alkan-2-ones andn-alkanes. This proposed mechanism for aqueousFTT synthesis differs from the surface-catalyzedstepwise FT process (i.e., gaseous) of polymerization of methylene reported in the literature.     

莱茵衣藻E2泛素结合酶CrUBC23调控油脂积累

【目的】为研究莱茵衣藻(Chlamydomonas reinhardtii)泛素结合酶(ubiquitin-conjugating enzymes,E2)CrUBC23在莱茵衣藻油脂代谢中的作用,为高产油微藻基因工程改良和揭示藻类油脂合成及代谢调控机理奠定基础。【方法】qRT-PCR分析莱茵衣藻在低氮、低磷胁迫下泛素结合酶CrUBC23表达情况;克隆CrUBC23同源基因干涉片段和全长基因,构建RNAi干涉载体和过量表达载体,转化莱茵衣藻并检测生物量和油脂含量;构建CrUBC23-GFP融合表达载体,用农杆菌浸染洋葱表皮细胞进行亚细胞定位。【结果】莱茵衣藻在低氮、低磷胁迫下CrUBC23基因表达量显著增加,增加幅度分别为正常培养的4.98–5.80倍和1.85–5.20倍。RNAi干扰结果显示,转基因藻细胞中性脂含量降低5.5%,总脂含量降低3.16%–17.6%。过量表达结果显示,转基因藻细胞中性脂含量增加8.8%,总脂含量增加4.51%–14.03%。【结论】CrUBC23正向调控莱茵衣藻油脂代谢,该基因定位于细胞核。     

Lipid composition and nitrogenase activity of symbioticFrankia (Alnus incana) in response to different oxygen concentrations

Summary The role ofFrankia vesicle envelope lipids in regulating oxygen diffusion of symbiotic nitrogen fixation inAlnus incana was examined. Total lipids of symbioticFrankia (vesicle clusters) that had been adapted to oxygen tensions of 5,21, or 40 kPa were analyzed with a normal phase HPLC system. During the oxygen treatment, nitrogenase activity was measured as hydrogen evolution in an open flow-through system. When plants were transferred to low oxygen (5 kPa) or high oxygen (40 kPa), nitrogenase activity dropped initially. Activity recovered in both treatments with a rate comparable to the controls (21 kPa O₂). Both lipid content and lipid composition of vesicle clusters were affected by the oxygen treatments. With increasing oxygen tension, the vesicle cluster lipid content increased. This correlated with structural data (fluorescence microscopy and TEM) which showed a thicker vesicle envelope at higher oxygen tension. Three hopanoid lipids, bacteriohopanetetrol (bht) and two isomers of phenylacetyl monoester of bht, made up approximately 80% of the vesicle cluster lipids. With changing oxygen concentrations, the ratio of the two bht esters changed whereas the relative proportion of bht remained fairly constant. Therefore, in theFrankia-Alnus incana symbiosis, adaptation to different ambient oxygen tensions occurs at least partly by increasing the thickness of theFrankia vesicle envelope and by changing its lipid composition.Abbreviations dw dry weight - bht bacteriohopanetetrol - SE standard error - TEM transmission electron microscopy Dedicated to the memory of Professor John G. Torrey     

Characterization of marine microalga, Scenedesmus sp. strain JPCC GA0024 toward biofuel production

A marine microalga, strain JPCC GA0024 was selected as high amount of neutral lipid producers from marine microalgal culture collection toward biofuel production. The strain was tentatively identified as Scenedesmus rubescens by 18S rDNA analysis. The growth of strain JPCC GA0024 was influenced by artificial seawater concentrations. The optimum growth of 0.79 g/l was obtained at 100% artificial seawater. The lipid accumulation reached 73.0% of dry cell weight at 100% artificial seawater without additional nutrients for 11 days. Gas chromatography/mass spectrometry analysis indicates that lipid fraction mainly contained hydrocarbons including mainly hexadecane (C₁₆ H₃₄) and 1-docosene (C₂₂ H₄₄). Furthermore, calorimetric analysis revealed that the energy content of strain JPCC GA0024 was 6,160 kcal/kg (25.8 MJ/kg) of calorific value, which was equivalent to the coal engery. The strain JPCC GA0024, S. rubescens, will become a promising resource that can grow as a dominant species in the seawater for the production of both liquid and solid biofuels.     

Tetraether membrane lipids of <Emphasis Type="Italic">Candidatus “Aciduliprofundum boonei”</Emphasis>, a cultivated obligate thermoacidophilic euryarchaeote from deep-sea hydrothermal vents

The lipid composition of Candidatus “Aciduliprofundum boonei”, the only cultivated representative of archaea falling in the DHVE2 phylogenetic cluster, a group of microorganisms ubiquitously occurring at hydrothermal vents, was studied. The predominant core membrane lipids in this thermophilic euryarchaeote were found to be composed of glycerol dibiphytanyl glycerol tetraethers (GDGTs) containing 0–4 cyclopentyl moieties. In addition, GDGTs with an additional covalent bond between the isoprenoid hydrocarbon chains, so-called H-shaped GDGTs, were present. The latter core lipids have been rarely reported previously. Intact polar lipid analysis revealed that they predominantly consist of GDGTs with a phospho-glycerol headgroup.     

耻垢分枝杆菌感染人脐带间充质干细胞模型的建立及其特征

【背景】结核分枝杆菌(Mycobacterium tuberculosis, Mtb)休眠菌形成被认为是潜伏结核感染(latent tuberculosis infection, LTBI)的主要原因,但目前缺乏体内和体外模型进行机制研究。新近研究表明Mtb可感染间充质干细胞(mesenchymal stem cells, MSC)并以休眠状态在细胞中长期存活。然而Mtb感染细胞模型存在周期长和生物安全要求高等问题,需要探索可用的MSC感染细胞模型用于Mtb休眠机制的研究。【目的】建立快速生长型耻垢分枝杆菌(Mycobacteriumsmegmatis,Ms)感染人脐带间充质干细胞(human umbilical cord MSC, hUCMSC)的细胞模型并研究其特征。【方法】取分离好的hUCMSC,流式细胞术鉴定其表面标志性抗原;以Ms菌株感染hUCMSC,DiI标记细胞膜,荧光显微镜下观察细胞吞噬作用;平板法计数Ms胞内存活率;油红O染色观察细胞脂滴形成;鬼笔环肽荧光染色观察细胞骨架变化;实时荧光定量PCR(realtimequantitativePCR,RT-qPCR)检测Ms...     

Seasonal patterns of total and energy reserve lipids of dominant zooplanktonic crustaceans from a hyper-eutrophic lake

Summary Seasonal patterns of lipid reserves and lipid classes of dominant zooplankton in a hyper-eutrophic lake were examined in relation to algal food resources. Triacylglycerol was the principle lipid energy reserve in all five species examined. During the height of the yearlyAphanizomenon flos-aquae bloom, lipid levels of the principle herbivores (Daphnia pulex andLeptodiaptomus sicilis) and an omnivore (Diacyclops bicuspidatus thomasi), were at their lowest concentration, suggesting that this cyanobacterium is nutritionally inadequate. As the cyanobacterial bloom began to collapse, bacterial numbers increased rapidly. The increase in bacterial numbers coincided with a large increase in areal lipid energy reserves ofDiaphanosoma leuchtenbergianum andChydorus sphaericus. Examination of seasonal patterns in the biomass of different algal species suggested thatRhodomonas minuta andCryptomonas erosa played a key role in nutrition, lipid deposition, and reproduction ofD. pulex andL. sicilis.