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.     

Growth and Osmoregulation of Chaetoceros muelleri in Relation to Salinity

The growth and osmoregulation of Chaetoceros muelleri Lemmermann(Bacillariophyceae) were investigated as a function of salinity.This centric diatom grew well over a wide range of salinityand required concentrations of NaCl above 10 mM for growth.Using gas chromatography- mass spectroscopy (GC-MS) analysisof cell extracts, we demonstrated that the alga contains anisomer of cyclohexanetetrol. The level of this isomer increasedwith increasing salinity. Levels of free amino acids also increasedwith increasing salinity, and quantitative determination withan amino acid analyzer revealed that the level of glutamic acidincreased the most with increases in salinity. Levels of intracellularK⁺ and Cl^– also increased significantly with increasesin salinity. Thus, in C. muelleri, not only organic solutessuch as the cyclohexanetetrol isomer and glutamic acid, butalso inorganic solutes such as K⁺ and Cl^– contribute toosmoregulation. (Received November 7, 1994; Accepted April 10, 1995)     

Effects of fundamental nutrient stresses on the lipid accumulation profiles in two diatom species Thalassiosira weissflogii and Chaetoceros muelleri

Bioprocess and Biosystems Engineering - Microalgae are considered as attractive feedstocks for biofuel production nowadays because of their high lipid contents and easy cultivation. In the present...     

Nitrite utilization by Chaetoceros muelleri under elevated CO2 concentration

Chaetoceros muelleri (Lemn.) was cultured with nitrite (NO₂⁻) or nitrate (NO₃⁻) as the sole nitrogen source and aerated with air or with CO₂-enriched air. Cells of C. muelleri excreted into the medium nitrite produced by reduction of nitrate when grown with 100 μM NaNO₃ as nitrogen source. Accordingly, NO₂⁻ concentration reached 10.4 μM after 95 h at the low CO₂ condition (aerated with air); while the maximum NO₂⁻ concentration was only around 2.0 μM at the high CO₂ condition (aerated with 5% CO₂ in air), furthermore, after 30 h it decreased to no more than 1.0 μM. NO₂⁻ was almost assimilated in 80 h when C. muelleri was cultured at the high CO₂ condition with 100 μM NaNO₂ as sole nitrogen source. At the high CO₂ condition, after 3 h the activity of nitrite reductase was as much as 50% higher than that at the low CO₂ condition. It was indicated that enriched CO₂ concentration could inhibit nitrite excretion and enhance nitrite assimilation by cells. Therefore, aeration with enriched CO₂ might be an effective way to control nitrite content in aquaculture systems.     

Overcoming shear stress of microalgae cultures in sparged photobioreactors

In the present work we identified and quantified the effect of hydrodynamic stress on two different microalgae strains, Dunaliella tertiolecta and D. salina, cultivated in bench-scale bubble columns. The cell death rate constant increased with increasing gas-entrance velocity at the sparger. Dunaliella salina was slightly more sensitive than D. tertiolecta. The critical gas-entrance velocities were approximately 50 and 30 m s(-1) for D. tertiolecta and D. salina, respectively. The effects of gas-flow rate, culture height, and nozzle diameter on the death rate constant were also studied. From these results it was concluded that bubble rising and bubble bursting are not responsible for cell death. Regarding nozzle diameter, small nozzles were more detrimental to cells. The bubble formation at the sparger was found to be the main event leading to cell death.     

Effects of shear flow on photosynthesis in a dilute suspension of microalgae

This study investigates the effects of shear stress on photosynthesis in dilute suspensions of Spirulina platensis and Chlorella by measuring the oxygen production rate using a coaxial, double-rotating-cylinder apparatus that generates Couette shear flow. Our device enables up to 0.6 Pa shear stress to be applied, which has the hydrodynamic effect of generating the algal motion and acutely augmenting the oxygen production rate of Spirulina, primarily because the surface area of algae exposed to illumination is increased. However, there is shear-flow limitation on any increase in oxygen production, and the shear stress at maximum oxygen production rate tends to decrease with increasing temperature. The comparative study with Chlorella showed the reverse relationship between oxygen production and shear stress, and the cause of this difference is discussed in terms of several factors such as size, shape, hydrodynamic stress capacity and others.     

The role of external carbonic anhydrase in photosynthesis during growth of the marine diatom Chaetoceros muelleri

Carbon dioxide concentrating mechanisms (CCMs) act to improve the supply of CO₂ at the active site of ribulose‐1,5‐bisphosphate carboxylase/oxygenase. There is substantial evidence that in some microalgal species CCMs involve an external carbonic anhydrase (CA_ext) and that CA_ext activity is induced by low CO₂ concentrations in the growth medium. However, much of this work has been conducted on cells adapted to air‐equilibrium concentrations of CO₂, rather than to changing CO₂ conditions caused by growing microalgal populations. We investigated the role of CA_ext in inorganic carbon (C_i) acquisition and photosynthesis at three sampling points during the growth cycle of the cosmopolitan marine diatom Chaetoceros muelleri. We observed that CA_ext activity increased with decreasing C_i, particularly CO₂, concentration, supporting the idea that CA_ext is modulated by external CO₂ concentration. Additionally, we found that the contribution of CA_ext activity to carbon acquisition for photosynthesis varies over time, increasing between the first and second sampling points before decreasing at the last sampling point, where external pH was high. Lastly, decreases in maximum quantum yield of photosystem II (F_v/F_m), chlorophyll, maximum relative electron transport rate, light harvesting efficiency (α) and maximum rates of C_i‐ saturated photosynthesis (V_max) were observed over time. Despite this decrease in photosynthetic capacity an up‐regulation of CCM activity, indicated by a decreasing half‐saturation constant for CO₂ (K_0.5CO₂), occurred over time. The flexibility of the CCM during the course of growth in C. muelleri may contribute to the reported dominance and persistence of this species in phytoplankton blooms.     

Culturing Chaetoceros muelleri using simplified media with different N sources: effects on production and lipid content

Land-based bivalve aquaculture depends on large-scale cultures of live microalgae for food. The intensity of large-scale microalgal production is important for cost-effectiveness. Using Walne’s medium as the control, simplified media containing nitrogen, phosphorus, silica, iron, manganese and vitamins were designed to determine the impact of nitrogen source and molar N:P ratio (sodium nitrate, NO₃ 9:1, ammonium chloride, NH₄ 9:1 and NH₄ 25:1) on growth, dry-weight biomass, culture longevity and lipid content of Chaetoceros muelleri, a diatom commonly used in shellfish aquaculture. During the exponential phase (day 6), dry-weight production in simplified media was similar to controls, indicating that this microalga can grow successfully on simplified media and use ammonium as the nitrogen source. The cultures grown on nitrate or ammonium differed in their time-course. Low nitrogen concentration in cultures grown with nitrate caused the collapse of these cultures within 11–13 days, after a short stationary phase. Cultures grown with ammonium had a longer stationary phase and were still alive on day 20, in spite of the low nitrogen concentrations observed after day 13 in cultures grown with NH₄ 9:1. During stationary phase (day 18) there was an increase in lipid content of algae under conditions of low nitrogen availability (NH₄ 9:1) and extended low phosphorus availability (NH₄ 25:1). Considering dry weight production, culture longevity, nutrient efficiency and lipid composition, simplified media containing ammonium, phosphorus, silica, iron, manganese and vitamins are a viable and profitable choice for batch culture of C. muelleri. In the exponential phase, the simplified medium NH₄ 9:1 was as effective as the control. Overall, both of the simplified media using ammonium are effective and suitable, depending on the purpose of the cultures and whether lipid contents (NH₄ 9:1), dry weight biomass (NH₄ 25:1) or nitrogen input and output (NH₄ 9:1) are desired.     

Effects of shear stress on articular chondrocyte metabolism

The articular cartilage of diarthrodial joints experiences a variety of stresses, strains and pressures that result from normal activities of daily living. In normal cartilage, the extracellular matrix exists as a highly organized composite of specialized macromolecules that distributes loads at the bony ends. The chondrocyte response to mechanical loading is recognized as an integral component in the maintenance of articular cartilage matrix homeostasis. With inappropriate mechanical loading of the joint, as occurs with traumatic injury, ligament instability, bony malalignment or excessive weight bearing, the cartilage exhibits manifestations characteristic of osteoarthritis. Breakdown of cartilage in osteoarthritis involves degradation of the extracellular matrix macromolecules and decreased expression of chondrocyte proteins necessary for normal joint function. Osteoarthritic cartilage often exhibits increased amounts of type I collagen and synthesis of proteoglycans characteristic of immature cartilage. The shift in cartilage phenotype in response to altered load yields a matrix that fails to support normal joint function. Mathematical modeling and experimental studies in animal models confirm an association between altered loading of diarthrotic joints and arthritic changes. Both types of studies implicate shear forces as a critical component in the destructive profile. The severity of cartilage destruction in response to altered loads appears linked to expression of biological factors influencing matrix integrity and cellular metabolism. Determining how shear stress alters chondrocyte metabolism is fundamental to understanding how to limit matrix destruction and stimulate cartilage repair and regeneration. At present, the precise biochemical and molecular mechanisms by which shear forces alter chondrocyte metabolism from a normal to a degenerative phenotype remain unclear. The results presented here address the hypothesis that articular chondrocyte metabolism is modulated by direct effects of shear forces that act on the cell through mechanotransduction processes. The purpose of this work is to develop critical knowledge regarding the basic mechanisms by which mechanical loading modulates cartilage metabolism in health and disease. This presentation will describe the effects of using fluid induced shear stress as a model system for stimulation of articular chondrocytes in vitro. The fluid induced shear stress was applied using a cone viscometer system to stimulate all the cells uniformly under conditions of minimal turbulence. The experiments were carried using high-density primary monolayer cultures of normal and osteoarthritic human and normal bovine articular chondrocytes. The analysis of the cellular response included quantification of cytokine release, matrix metalloproteinase expression and activation of intracellular signaling pathways. The data presented here show that articular chondrocytes exhibit a dose- and time-dependent response to shear stress that results in the release of soluble mediators and extracellular matrix macromolecules. The data suggest that the chondrocyte response to mechanical stimulation contributes to the maintenance of articular cartilage homeostasis in vivo.     

Sustainable, High-Yielding Outdoor Mass Cultures of Chaetoceros muelleri var. subsalsum and Isochrysis galbana in Vertical Plate Reactors

Continuous cultures of Chaetoceros muelleri and Isochrysis galbana were grown outdoors in flat plate-glass reactors in which light-path length (LPL) varied from 5 to 30 cm. High daily productivity (13 to 16 g cell mass per square meter of irradiated reactor surface) for long periods of time was obtained in reactors in which the optical path as well as cell density were optimized. 'Twenty centimeters was the optimal LPL, yielding the highest areal productivity of cell mass (g m^–2d^–1), eicosapentaenoic acid, and docosahexaenoic acid, which was identical with that previously found for polysaccharide production of Porphyridium and not far from the optimal LPL affecting maximal productivity in Nannochloropsis species. Relating the energy impinging on a given reactor surface area to the appropriate number of cells showed that the most efficient light dose per cell, obtained with the 20-cm LPL reactor, was approximately 2.5 times lower than the light dose available per cell in the 5-cm LPL reactor, in which a significant decline in areal cell density accompanied the lowest areal output of cell mass. The most effective harvesting regimen was in the range of 10% to 15% of culture volume harvested daily and replaced with fresh growth medium, resulting in a sustainable culture density of 24 × 10⁶ and 28 × 10⁶ cells/ml of C. muelleri and I. galbana, respectively.     

Effects of shear stress on endothelial cell haptotaxis on micropatterned surfaces

Endothelial cell (EC) migration plays a critical role in vascular remodeling. Here we investigated the interactions between haptotaxis (induced by extracellular matrix gradient) and mechanotaxis (induced by mechanical forces) during EC migration. A micropatterning technique was used to generate step changes of collagen surface density. Due to haptotaxis, ECs developed focal adhesions and migrated into the area with higher surface density of collagen. Different levels of fluid shear stress were applied on ECs in the direction perpendicular to collagen strips. Shear stress at 2 dyn/cm2 did not affect haptotaxis, while shear stress at 3 dyn/cm2 or higher was sufficient to drive the migration of most ECs in the flow direction and against haptotaxis. Immunostaining revealed the increase of focal adhesions and lamellipodial protrusion in the direction of flow. These results suggest that shear stress beyond a certain threshold can be a predominant factor to determine the direction of EC migration.     

Effects of luminal shear stress on cerebral arteries and arterioles

The effect of luminal shear stress was studied in cerebral arteries and arterioles. Middle cerebral arteries (MCA) and penetrating arterioles (PA) were isolated from male Long-Evans rats, mounted in a tissue bath, and pressurized. After the development of spontaneous tone, inside diameters were 186 +/- 5 microm (n = 28) for MCA and 65 +/- 3 microm (n = 37) for PA. MCA and PA constricted approximately 20% with increasing flow. Flow-induced constriction persisted in MCA and PA after removal of the endothelium. After removal of the endothelium, the luminal application of a polypeptide containing the Arg-Gly-Asp amino acid sequence (inhibitor of integrin attachment) abolished the flow-induced constriction. Similarly, an antibody specific for the beta(3)-chain of the integrin complex significantly inhibited the flow-induced constriction. The shear stress-induced constriction was accompanied by an increase in vascular smooth muscle Ca(2+). For example, a shear stress of 20 dyn/cm(2) constricted MCA 8% (n = 5) and increased Ca(2+) from 209 +/- 17 to 262 +/- 29 nM (n = 5). We conclude that isolated cerebral arteries and arterioles from the rat constrict to increased shear stress. Because the endothelium is not necessary for the response, the shear forces must be transmitted across the endothelium, presumably by the cytoskeletal matrix, to elicit constriction. Integrins containing the beta(3)-chain are involved with the shear stress-induced constrictions.     

Mechanical stress tolerance of two microalgae

Aim of the present work is quantifying the mechanical stress generated by some major process equipment used in massive microalgae culturing plants (centrifugal and air-lift pumps, and nozzles) and highlighting its effects on the microalgal population. Two microalgal species were used as test cases: Chlorella vulgaris (unicellular) and Scenedesmus dimorphus 1237 (colonial). The evaluation of the shear effect on algal growth was carried out through measurement of absorbance, photosynthetic activity (oxygen evolution) and variable chlorophyll fluorescence. Cell aggregate development/breakage was effectuated by visual inspection and light scattering. The use of centrifugal pumps for culture recycling strongly affected the growth of C. vulgaris, while nozzles effects were confined to aggregate breakage of S. dimorphus. The analysis of experimental data is supported by the consideration of hydrodynamic stress calculated by: shear rate, shear stress, stress volumes/times, energy dissipation rates, and turbulence microscale size.     

Effects of small-scale turbulence on microalgae

Turbulence flows are characterized by their viscous dissipation rates ? and the kinematic viscosity of the fluid ν, but the effects of turbulence on organisms such as microalgae smaller than the Kolmogorov inertial-viscous length scale L_K ≡ (ν³/ε)^/14 depend on the stress τ ≡ µγ, where µ = ?ν is the dynamic viscosity, ρ is the density, and the rate-of-strain γ ≡ (ε/ν)^/12. While various workers have shown qualitatively that turbulence affects several microalgal physiological processes, these effects have not been quantified in terms of ε, τ or γ. Various microalgal groups seem to have different sensitivities to inhibition by turbulence. The relative sensitivities aregreen algae < blue-green algae < diatoms < dinoflagellates with dinoflagellates being most sensitive. We have quantified the growth sensitivity to turbulence for a red tide dinoflagellate,Gonyaulax polyedra Stein, by imposing constant γ values on cultures placed within a gap between rotating outer and fixed inner concentric cylinders. Threshold turbulence values for growth inhibition are consistent with turbulence parameters near the sea surface with light winds, suggesting turbulence may be the reason that high winds inhibit red tides. For ? > 0.18 cm²s^?3, τ > 0.04 dynes cm^?2 (0.002 N M^?2 or Pa), γ > 4.4 rad s^?1, cell numbers and chlorophyll fluorescence declined, and cells lost their longitudinal flagella and the ability to swim forward. At lower ε, τ and γ values growth rates and cell morphology were the same as in unsheared control cultures. High turbulence may affect other algae, such asSpirulina, which are commonly mass cultured.     

Effects of shear stress on differentiation of stem cells into endothelial cells

Stem cell transplantation is an appealing potential therapy for vascular diseases and an indispensable key step in vascular tissue engineering. Substantial effort has been made to differentiate stem cells toward vascular cell phenotypes, including endothelial cells (ECs) and smooth muscle cells. The microenvironment of vascular cells not only contains biochemical factors that influence differentiation but also exerts hemodynamic forces, such as shear stress and cyclic strain. More recently, studies have shown that shear stress can influence the differentiation of stem cells toward ECs. A deep understanding of the responses and underlying mechanisms involved in this process is essential for clinical translation. This review highlights current data supporting the role of shear stress in stem cell differentiation into ECs. Potential mechanisms and signaling cascades for transducing shear stress into a biological signal are proposed. Further study of stem cell responses to shear stress will be necessary to apply stem cells for pharmacological applications and cardiovascular implants in the realm of regenerative medicine.     

Effects of biaxial oscillatory shear stress on endothelial cell proliferation and morphology

Wall shear stress (WSS) on anchored cells affects their responses, including cell proliferation and morphology. In this study, the effects of the directionality of pulsatile WSS on endothelial cell proliferation and morphology were investigated for cells grown in a Petri dish orbiting on a shaker platform. Time and location dependent WSS was determined by computational fluid dynamics (CFD). At low orbital speed (50 rpm), WSS was shown to be uniform (0-1 dyne/cm(2)) across the bottom of the dish, while at higher orbital speed (100 and 150 rpm), WSS remained fairly uniform near the center and fluctuated significantly (0-9 dyne/cm(2)) near the side walls of the dish. Since WSS on the bottom of the dish is two-dimensional, a new directional oscillatory shear index (DOSI) was developed to quantify the directionality of oscillating shear. DOSI approached zero for biaxial oscillatory shear of equal magnitudes near the center and approached one for uniaxial pulsatile shear near the wall, where large tangential WSS dominated a much smaller radial component. Near the center (low DOSI), more, smaller and less elongated cells grew, whereas larger cells with greater elongation were observed in the more uniaxial oscillatory shear (high DOSI) near the periphery of the dish. Further, cells aligned with the direction of the largest component of shear but were randomly oriented in low magnitude biaxial shear. Statistical analyses of the individual and interacting effects of multiple factors (DOSI, shear magnitudes and orbital speeds) showed that DOSI significantly affected all the responses, indicating that directionality is an important determinant of cellular responses.     

CO2对牟氏角毛藻高密度培养的影响

以牟氏角毛藻为材料,通过对比实验研究了CO2和CO2通入方式对光合生物反应器中高密度培养的牟氏角毛藻的生长速度和细胞密度的影响;并以水体pH值为指标,控制CO2的通入数量,进而研究了牟氏角毛藻在我们自行研制的卧式光合生物反应器中的生长情况.结果表明细胞悬浮培养达到一定密度之后,CO2的供应,可以有效地提高生长速度和细胞密度.对CO2通入方式的实验结果表明,不同的CO2通入方式,产生的效果不同,其中以空气和纯CO2混合后通入,效果最好.     

CO_2对牟氏角毛藻高密度培养的影响

以牟氏角毛藻为材料,通过对比实验研究了CO2和CO2通入方式对光合生物反应器中高密度培养的牟氏角毛藻的生长速度和细胞密度的影响;并以水体pH值为指标,控制CO2的通入数量,进而研究了牟氏角毛藻在我们自行研制的卧式光合生物反应器中的生长情况。结果表明细胞悬浮培养达到一定密度之后,CO2的供应,可以有效地提高生长速度和细胞密度。对CO2通入方式的实验结果表明,不同的CO2通入方式,产生的效果不同,其中以空气和纯CO2混合后通入,效果最好。