Mathematical model of Chlorella minutissima UTEX2341 growth and lipid production under photoheterotrophic fermentation conditions

To reduce the cost of algal biomass production, mathematical model was developed for the first time to describe microalgae growth, lipid production and glycerin consumption under photoheterotrophic conditions based on logistic, Luedeking-Piret and Luedeking-Piret-like equations. All experiments were conducted in a 2 L batch reactor without considering CO(2) effect on algae's growth and lipid production. Biomass and lipid production increased with glycerin as carbon source and were well described by the logistic and Luedeking-Piret equations respectively. Model predictions were in satisfactory agreement with measured data and the mode of lipid production was growth-associated. Sensitivity analysis was applied to examine the effects of certain important parameters on model performance. Results showed that S(0), the initial concentration of glycerin, was the most significant factor for algae growth and lipid production. This model is applicable for prediction of other single cell algal species but model testing is recommended before scaling up the fermentation of process.     

Watching intracellular lipolysis in mycobacteria using time lapse fluorescence microscopy

The fact that Mycobacterium tuberculosis mobilizes lipid bodies (LB) located in the cytosol during infection process has been proposed for decades. However, the mechanisms and dynamics of mobilization of these lipid droplets within mycobacteria are still not completely characterized. Evidence in favour of this characterization was obtained here using a combined fluorescent microscopy and computational image processing approach. The decrease in lipid storage levels observed under nutrient depletion conditions was correlated with a significant increase in the size of the bacteria. LB fragmentation/condensation cycles were monitored in real time. The exact contribution of lipases in this process was confirmed using the lipase inhibitor tetrahydrolipstatin, which was found to prevent LB degradation and to limit the bacterial cell growth. The method presented here provides a powerful tool for monitoring in vivo lipolysis in mycobacteria and for obtaining new insights on the growth of cells and their entry into the dormant or reactivation phase. It should be particularly useful for studying the effects of chemical inhibitors and activators on cells as well as investigating other metabolic pathways.     

Characterization of the growth of 2D protein crystals on a lipid monolayer by ellipsometry and rigidity measurements coupled to electron microscopy.

We present here some sensitive optical and mechanical experiments for monitoring the process of formation and growth of two-dimensional (2D) crystals of proteins on a lipid monolayer at an air-water interface. The adsorption of proteins on the lipid monolayer was monitored by ellipsometry measurements. An instrument was developed to measure the shear elastic constant (in plane rigidity) of the monolayer. These experiments have been done using cholera toxin B subunit (CTB) and annexin V as model proteins interacting with a monosialoganglioside (GM1) and dioleoylphosphatidylserine (DOPS), respectively. Electron microscopy observations of the protein-lipid layer transferred to grids were systematically used as a control. We found a good correlation between the measured in-plane rigidity of the monolayer and the presence of large crystalline domains observed by electron microscopy grids. Our interpretation of these data is that the crystallization process of proteins on a lipid monolayer passes through at least three successive stages: 1) molecular recognition between protein and lipid-ligand, i.e., adsorption of the protein on the lipid layer; 2) nucleation and growth of crystalline patches whose percolation is detected by the appearance of a non-zero in-plane rigidity; and 3) annealing of the layer producing a slower increase of the lateral or in-plane rigidity.     

The effect of growth temperature on the membrane lipid environment of the psychrophilic bacterium Micrococcus cryophilus

The relationship between the delta 9-desaturase activity of the psychrophilic bacterium Micrococcus cryophilus grown at different temperatures and the physical state of its membrane lipids as measured by ESR spectroscopy has been studied. Arrhenius plots of desaturase activity were biphasic with a discontinuity at a temperature which depended upon the bacterial growth temperature. Changes in the desaturase activation energy, which increased as the growth temperature was lowered, are discussed in the context of membrane lipid fluidity adaptation to changing environmental temperature. The fluidity of membranes and isolated lipids was measured using nitroxide-labeled fatty acids. The spectra of 2-(10-carboxydecyl)-2-hexyl-4,4-dimethyl-3-oxazolidinoxyl in membranes indicated that there were two lipid environments within the membrane whose relative proportions were dependent both on temperature of measurement and on bacterial growth temperature. In contrast, 2-(3-carboxypropyl)-4,4-dimethyl-2-tridecyl-3-oxazolidinoxyl spectra showed a single lipid environment and plots of log order parameter (S3) vs 1/T were biphasic with inflexion temperatures which were closely related to the bacterial growth temperature. As with membranes, plots of log S3 vs 1/T for total lipids, phosphatidylglycerol and cardiolipin, but not phosphatidylethanolamine, were biphasic and showed inflexions which correlated well with bacterial growth temperature. These results are interpreted as being consistent with a location for the desaturase within the bulk lipid of the membrane rather than in association with specific lipid types.     

长孢被孢霉利用淀粉质原料发酵产微生物油脂

通过研究长孢被孢霉(Mortierella elongate)的发酵过程,并采用摇瓶分批补料发酵模式考察了起始补料时间及补料基质对长孢被孢霉合成微生物油脂的影响。结果发现该菌株合成油脂主要在发酵48~96 h进行,且N源对菌株的生长有促进作用,采用限氮补料发酵可大幅度提高微生物油脂的产量。最适培养条件:可溶性淀粉20 g/L,玉米浆3 g/L,补料起始时间为发酵48 h,单次补加可溶性淀粉4 g。在此条件下,油脂产量较不补料时增加了521.74 mg,增长率为237.1%。     

The effect of growth inhibiting factors on the total lipid content of the zebrafish, Brachydanio rerio (Hamilton-Buchanan)

The effects of growth inhibiting factors on the total lipid content of zebrafish was studied. Crowded zebrafish were subjected to extraction of the aquarium water using activated charcoal. Control fish were crowded, but were not subjected to extraction. This resulted in a gain in mean wet weight of 10.9% for the crowded, extracted fish (experimentals) as compared to the controls. In order to determine the effects of crowding on the total lipid content of the crowded fish, lipid analyses were conducted at the conclusion of the growth studies. These studies indicate that the fish in the extracted system had a greater lipid content than those in the-non-extracted system (15.4% v. 10.4% mean lipid). It is proposed that growth inhibiting factors libeerated from the crowded fish may affect the thyroid gland, causing lipid mobilization from the tissues resulting in resulting in lowered total lipid in these animals. Extraction of the aquarium water with activated charcoal removes the growth inhibiting factor, possibly decreasing thyroid function with a resulting higher total lipid content.     

The relationships between corpora allata and fat body and haemolymph lipids in the adult female desert locust

Allatectomy of 1-day-old female desert locusts resulted in an accumulation of lipid in the fat body. This accumulation of lipid was due to the continuation of lipid deposition in the fat body after the period of somatic growth. Somatic growth and feeding activity were unaffected by allatectomy, and so could not be indirect causes of fat body lipid accumulation. Lipid accumulation in allatectomized locusts is more likely to be related directly to a lack of juvenile hormone. Implantation of active corpora allata into 1-day-old adult female locusts resulted in a premature development of oöcytes and a decrease in fat body lipid accumulation; somatic growth was not inhibited. Implantation of active corpora allata into old allatectomized locusts resulted in a decrease in the fat body lipid content and the onset of oöcyte development. The lipid synthetic activity of the fat body, measured by the incorporation of ¹⁴C-actate into total fat body lipid, was greatly increased in allatectomized locusts after the period of somatic growth. The protein synthetic activity of the fat body, measured by the incorporation of ³H-leucine into total fat body protein, remained low after the period of somatic growth in allatectomized insects. Juvenile hormone might thus have a dual effect on fat body metabolism, that is suppressing lipid synthesis and stimulating vitellogenic protein synthesis. Increased synthesis of lipid by the fat body would then account for the accumulation of lipid in the fat body after allatectomy. Inhibition of release of lipid from the fat body is unlikely to play a part in the accumulation as allatectomy had no effect on haemolymph lipid concentrations.     

Stimulation of lymphocytes by concanavalin A. Temperature-dependent effect of fatty acid replacements

The membrane fatty acyl composition of lymphocytes was altered by growth in lipid-depleted serum containing fatty acid supplements, as well as avidin to block endogenous synthesis of fatty acids. Under these growth conditions over 50% of the total fatty acid in membrane phospholipid were derived from the added fatty acid. Enrichment of lymphocyte membranes with oleate (cis C18:1) or elaidate (trans C18:1) shifted the optimum temperature for mitogenic stimulation by concanavalin A as measured by [3H]thymidine incorporation. These results suggest that the fluidity of the membrane lipid phase plays a role in the process of lymphocyte stimulation by lectins.     

Prediction of lipid accumulation-degradation in oleaginous micro-organisms growing on vegetable oils

Oleaginous micro-organisms (yeasts, moulds), in culture media having the carbon source as limited factor, degrade reserve lipids and produce new biomass, after the onset of carbon exhaustion from the medium. In this paper the process of lipid accumulation-degradation in oleaginous micro-organisms, growing on a vegatable oil was simulated. The model was integrated with 4 different methods and the parameters were optimised with the least squares method. It was found that the degradation of endocellular carbon pool is a very slow process characterised, however, by a good yield in fat-free biomass. Low values of the specific growth rates of the fat-free microbial mass, both from consumption of extra cellular and endocellular carbon pools, favourite the production of microbial lipid. The maximum of the specific rate of lipid accumulation is positively affected by the low values of the specific growth rate of the fat-free microbial mass from consumption of extra cellular carbon pool, but remained unaffected by the specific growth rate of the fat-free microbial mass from consumption of endocellular carbon pool. On the other hand, lipid production and specific rate of lipid accumulation are positively influenced by the high values of the specific rate of storage lipid formation. In conclusion, this numerical model can be used in the laboratory as pilot for planing further experimental work.     

Tetraether type polar lipids increase after logarithmic growth phase of Methanobacterium thermoautotrophicum in compensation for the decrease of diether lipids

Abstract The ratios of tetraether to diether type lipids in the total lipid during cell growth in batch cultures of Methanobacterium thermoautotrophicum ΔH (DSM 1053) were examined. The proportion of tetraether type lipids to the total lipid was about 80% during the log phase, and at the onset of the transient phase it began to rise up to about 93%. It was kept almost constant at that level throughout the stationary phase. The polar lipid composition changed with the age of the cell culture. The proportions of all the diether type polar lipids were lower and the levels of all tetraether type polar lipids were higher in the stationary phase than in the log phase. On the other hand, the composition of polar head groups, irrespective of the core lipids, was nearly constant in both growth phases measured so far despite the change in core lipid composition.     

Response surface methodology as a decision-making tool for optimization of culture conditions of green microalgae Chlorella spp. for biodiesel production

We have evaluated process optimization and the interactive effects of a number of variables using a Box–Behnken design of response surface methodology (RSM). The process variables nitrate, phosphate, glucose and pH were optimized to enhance the cell growth rate, lipid accumulation and other biochemical parameters of Chlorella spp. The most significant increase in lipid production (dry cell weight basis) occurred at limited concentrations of nitrate and phosphate, 1 % glucose and pH 7.5. The addition of nitrates during the mid-lag and mid-exponential phases produced the maximum inhibitory effect on lipid accumulation and the presence of yeast extract led to a further enhancement of lipid accumulation. Of all the media tested, BG-11 was the best suited medium for algal biomass production and chlorophyll content. A significant increase in algal biomass was observed in BG-11 supplemented with bicarbonate and glucose (1 %). The maximum specific growth rate observed was on 9th day of culturing. Results of optimization of process variables through response surface methodology and optimization of various other conditions reflect cutting edge research directed towards increasing algal biomass and lipid content for biodiesel production using an efficient economical technological approach.     

Characterization of the growth and lipid content of the diatom Chaetoceros muelleri

Chaetoceros muelleri (Schütt) was cultured on a thermal gradient plate, subjected to two media types with a range of specific conductances, and evaluated for growth and neutral lipid accumulation. Growth was measured directly by daily changes in cell numbers and indirectly by changes in optical density at 750 nm. C. muelleri exhibited a growth rate of at least two doublings day-1 over broad temperature (20 to 35 °C) and conductance ranges (10 to over 60 mS cm-1) and the optimum growth rate approached 4.0 doublings day-1 at 30 °C and a conductance of 25 mS cm-1. Intracellular neutral lipid storage was evaluated with fluorometry and epifluorescent microscopy using the fluorochrome Nile Red. Gravimetric analysis revealed a total lipid content in nitrogen-depleted cultures of C. muelleri of over 400 mg L-1, five to seven times that observed in nitrogen-replete cultures. Based on its high growth rate, tolerance to a broad range of temperatures and specific conductances, and large quantity of intracellular lipid, C. muelleri may have potential for exploitation as a renewable precursor to liquid fuels or as a lipid source. This revised version was published online in August 2006 with corrections to the Cover Date.     

Liposomal forms of rhenium cluster compounds: enhancement of biological activity

Liposomal formulations of dinuclear cluster rhenium (Re) compounds were used in biochemical trials. Interaction of liposomal forms of some Re compounds with red blood cells in experiments in vitro showed strong cell-stabilizing properties. In the models of tumor growth and hemolytic anemia in vivo, liposomal forms had better therapeutic effects in comparison with their solutions. The process of formation of liposomes of cluster Re compounds with different organic ligands was investigated by the method of electronic absorption spectra and mechanism of their interactions with lipids is proposed. Encapsulation of cluster Re compounds to lipid coating may have activation significance for the quadruple Re-Re bond.     

耐乙酸粘红酵母合成油脂

乙酸是木质纤维素在水解过程中的主要副产物,高浓度的乙酸严重影响产油微生物的生长和油脂合成。本文研究了粘红酵母对乙酸的耐受性及其利用乙酸合成微生物油脂的能力。结果表明,在初始葡萄糖、木糖浓度分别为6 g/L和44 g/L的混合糖培养基中,乙酸浓度低于10 g/L时,不会对菌体生长产生抑制作用,油脂合成还得到了促进。当乙酸添加量为10 g/L时,生物量、油脂产量、油脂含量较对照组分别提高了21.5%、171.2%和121.6%。进一步研究表明,粘红酵母具备利用乙酸合成油脂的能力,当以乙酸为唯一碳源,浓度为25 g/L时,油脂产量达到3.20 g/L,油脂质量得率为13%。微生物油脂成分分析表明,粘红酵母以乙酸为底物制得的油脂可以作为制备生物柴油的油脂原料,其主要成分为棕榈酸、硬脂酸、油酸、亚油酸和亚麻酸,其中饱和脂肪酸和不饱和脂肪酸含量分别为40.9%和59.1%。由于粘红酵母具有利用乙酸合成微生物油脂的能力,在以木质纤维素水解液为原料生产微生物油脂的脱毒过程中,一定浓度的乙酸可以不必脱除。     

Composition and synthesis of cellular lipids in Neurospora crassa during cellular differentiation.

The synthesis of cellular lipids of Neurospora crassa was measured during growth on low (2% sucrose)- and high (15% glucose)-carbohydrate supplementation. The amount of lipid per dry weight of cells does not change during the germination and early logarithmic growth periods, but the percentage of phospholipid in the lipid does increase, reaching a maximal value of 90% at 4 to 5 h after inoculation, at which time the phospholipid content of the cells is approximately 60 mumol/g (dry weight). The content of the anionic phospholipids, as a percentage of the lipid fraction, is relatively constant during the growth period, but the contents of the zwitterionic phospholipids phosphatidylcholine and phosphatidylethanolamine change in a reciprocal fashion. During the first 8 h of growth, phosphatidylcholine falls from 53% of the phospholipid to 43%, whereas phosphatidylethanolamine rises from 29 to 38%. The total of these two phospholipids is approximately 83% during the growth period studied. The synthesis of cellular phospholipids, measured either by [32P]H3PO4 or [14C]glucose incorporation, reached maximal levels between 3 and 5 h of growth. The effect of the high-carbohydrate supplement on cellular lipids was minimal. Inclusion of 15% glucose decreased the labeling of phospholipid by [32P]H3PO4, but did not affect lipid composition. This observation is in contrast to the effects of high glucose on mitochondrial phospholipid synthesis.     

Thyrotropin regulation of membrane lipid fluidity in the FRTL-5 thyroid cell line. Its relationship to cell growth and functional activity

The mitogenic effect of thyrotropin on functional rat thyroid cells of the line FRTL-5 is correlated with membrane lipid fluidity as evaluated by fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene. Continued exposure of FRTL-5 cells to a medium lacking thyrotropin causes cessation of cell proliferation and a decrease in membrane lipid fluidity which reaches its minimum in approximately 8 days. The change in lipid fluidity is due to an absolute increase (greater than 2-fold) of membrane cholesterol, with an increased cholesterol/phospholipid ratio and an increased ratio of saturated to unsaturated fatty acids of the membrane phospholipids, contributed primarily by a nearly 4-fold increase in the ratio of saturated to unsaturated C16 fatty acids. It is also associated with a variation of the relative proportions of the major membrane phospholipids; thus, phosphatidylinositol and phosphatidylethanolamine decrease while phosphatidylcholine increases. Both membrane fluidity and lipid composition can be restored by thyrotropin to their original levels, i.e. levels measured under continuous exposure to the hormone. Complete reversal requires at least 48 h, i.e. approximately the same time required for resumption of growth when FRTL-5 cells, starved in thyrotropin, are re-exposed to the hormone. Changes in lipid composition and fluidity can be prevented or can be reversed if FRTL-5 cells are exposed to dibutyryl cAMP while being deprived of thyrotropin. Dibutyryl cAMP has only a modest direct effect on growth; however, this pretreatment eliminates the 48-h lag phase with respect to thyrotropin stimulation. It is proposed that the effects of thyrotropin on growth of FRTL-5 cells requires a modification of the molecular structure and the physical state of cell membranes, which can be mediated by cAMP, although cAMP is not sufficient by itself to promote growth.     

Effect of liposomal antitumor preparation 5,6-benzocoumarin-5-uracil on intensity of free-radical processes in the liver microsomes and tumor cells of rats with transplanted Guerin's carcinoma

The contents of primary and secondary (TBA-active) products of lipid peroxidation were investigated in microsomal fraction of the liver and tumor cells of rats with transplanted Guerin's carcinoma and under the condition of antitumor liposomal preparation 5,6-benzcumarine-5-uracil (BCU) action. High level of lipid peroxidation process in the microsomal fraction is shown in the rat liver and tumor cells under the condition of BCU action in the period of intensive carcinoma growth. It remains till the period of tumor growth braking. This fact testifies to the prooxidation action of the preparation. Liposomal antitumor preparation BCU raises the process of lipid peroxidation in microsomal fraction of tumor cells and its action increases according to the malignant growth. The processes of lipid peroxidation in microsomal rat liver fraction approach the control data under the condition of the mentioned preparation. The investigated liposomal form of BCU possesses directed prooxidation action on the malignant tissue.     

粘红酵母和酿酒酵母联合处理味精废水

为了解决味精废水中高NH4+浓度抑制油脂微生物的生长和油脂积累问题,采用粘红酵母和酿酒酵母联合处理味精废水的方法:首先利用酿酒酵母降解味精废水(MSG)中NH4+,然后将处理后的废水进一步发酵培养合成油脂。研究结果表明:用经酿酒酵母预处理过的味精废水作为粘红酵母的培养基发酵时,粘红酵母的生物量为33.3 g/L,油脂产率为18.16%,COD降解率为50.6%,NH4+的降解率为93.9%。比粘红酵母单独处理味精废水,NH4+的降解率提高了6.14倍,生物量、油脂产率和COD降解率分别提高了8.1%、30.06%和9.58%。     

Factors affecting lipid accumulation by <Emphasis Type="Italic">Nannochloropsis oculata</Emphasis> in a two-stage cultivation process

Reserve lipids of microalgae are promising for biodiesel production. However, optimization of cultivation conditions for both biomass yield and lipid production of microalgae is a contradictory problem because required conditions for both targets are different. In this study, a two-stage cultivation strategy is proposed to enhance lipid production of the microalga Nannochloropsis oculata. Biomass growth and lipid production were carried out in two separate and non-interacting stages. In first-stage cultivation, microalgae were cultivated in optimal conditions for cell growth. Then, microalgae were harvested and transferred into a growth-limited environment, thus enhancing lipid production of microalgae. Here, optimization of the lipid production stage (second stage) with respect to different levels of inoculum concentration, salinity of culture broth, and intensity of irradiance was performed. The results show that irradiance exhibits a significant influence on lipid production. The highest lipid productivity of 0.324 g L⁻¹ day⁻¹ was obtained with an inoculum concentration of 2.3 g L⁻¹, a salinity of 35 g L⁻¹, and an irradiance of 500 μmol photons m⁻² s⁻¹. The final yield of lipid obtained from the two-stage process was 2.82-times higher than that from traditional single-stage batch cultivation systems.     

Optimisation of docosahexaenoic acid production in batch cultivations by Crypthecodinium cohnii

The heterotrophic micro alga Crypthecodinium cohnii was cultivated in media containing glucose, yeast extract and sea salt. Increasing amounts of yeast extract stimulated growth but influenced lipid accumulation negatively. Sea salt concentrations above half the average seawater salinity were required for good growth and lipid accumulation. C. cohnii was able to grow on a glucose concentration as high as 84.3 g l⁻¹, although concentrations above 25 g l⁻¹ decreased the growth rate. Comparison of growth at 27 and 30°C showed that the higher incubation temperature was more favourable for growth. However, lipid accumulation was higher at the lower incubation temperature. In a bioreactor the biomass concentration increased from 1.5 to 27.7 g l⁻¹ in 74 h. In the final 41 h of the process the lipid content of the biomass increased from 7.5 to 13.5%. In this period the percentage of docosahexaenoic acid of the lipid increased from 36.5 to 43.6%. The total amounts of lipid and docosahexaenoic acid after 91 h were 3.7 and 1.6 g l⁻¹, respectively.