Hydrodynamic shear breakage of DNA

The rate of breakage of duplex DNA molecules by laminar flow through a capillary has been studied. For λb2b5c DNA (molecular wt., M = 25 × 10⁶) the point at which breakage occurs is normally distributed around the center of the molecule with a standard deviation of 12.5% of the molecular length. At constant shear stress or shear rate, the breakage rate is independent of ionic strength. Thus, shear induced local denaturation is not a rate limiting, preliminary step in breakage. In experiments at constant temperature with varying solvent viscosity (controlled by added sucrose) the breakage rate is a function of shear rate, not of shear stress. The rate of opening of hydrogenbonded circles into linear molecules by hydrodynamic shear is also shown to be a function of shear rate and not of shear stress. The breakage rate at constant shear rate is not greatly dependent on temperature. The shear rate required to achieve breakage is inversely proportional to M^1,2. The breakage rate constant, k varies as a very high power of the shear rate; at 25°C, d In k/d In Gm ～ 15; at 10°C, d In k/d In Gm ～ 26, where Gm is the maximum shear rate at the capillary wall. The unexpected result that breakage rate is mainly dependent on shear rate, not shear stress, supports a model in which the DNA molecule is distorted with a driving force which depends on the hydrodynamic shear stress, ηG, but the rate limiting step is segment diffusion into a highly extended configuration. The characteristic time to achieve this configuration is proportional to solvent viscosity, η, hence the breakage rate is dependent on ηG/η or G, the shear rate.     

Production of antifungal saponins in an airlift bioreactor with a cell line transformed from Solanum chrysotrichum and its activity against strawberry phytopathogens

Abstract

Biotechnology through plant cell cultures in bioreactors is a tool that allows increasing the production of secondary metabolites of commercial interest. The hydrodynamic characterization, in addition to the transfer (OTR) and uptake (OUR) of oxygen through the dynamic method with different aeration rate, were used to see their influence on the production of biomass and saponins. The culture poisoning technique was used to determine the antifungal activity of the SC-2 and SC-3 saponins in vitro. Likewise, the shear or hydrodynamic stress of 273.6?mN/m² were calculated based on the Reynolds Number. The oxygen supply (OTR) was always greater than the demand (OUR) for all the aeration rate evaluated. Dry weight values of 8.6 gDW/L and a concentration of 2.7?mg/L and 187.3?mg/L of the saponins SC-2 and SC-3 respectively were obtained with an air flow of 0.1 vvm. In addition, it was possible to inhibit the growth of phytopathogenic fungi in vitro by up to 93%, while in vivo it was possible to reduce the infections of strawberry seeds inoculated with phytopathogens, obtaining up to 94% of germinated seeds. This information will facilitate the rational operation of the bioreactor culture system that produces secondary metabolites.     

Production and nutritional quality of the rotifer Brachionus plicatilis fed marine Chlorella sp. at different cell densities

The effect of different cell densities of marine Chlorella sp. on the growth rate, doubling time and production of the rotifer Brachionus plicatilis was investigated. A significant increase in rotifer production was achieved at a density of 50 × 10⁶ Chlorella cells ml^–1. The nutritional quality of rotifers grown at different concentrations of Chlorella is discussed.     

Influence of culture vessel characteristics and agitation rate on gaseous exchange,hydrodynamic stress,and growth of embryogenic cork oak (<Emphasis Type="BoldItalic">Quercus suber</Emphasis> L.) cultures

Somatic embryogenesis can be induced in the leaves of cork oak (Quercus suber L.) trees. The use of this propagation system in multivarietal forestry requires the mass production of cloned plants at low cost. Investigations were made into the influence of three types of Erlenmeyer flask and three orbiting speeds (60, 110, and 160 rpm) on oxygen transfer rate (K_L a), the shear force index (SFI), biomass production, and the proliferation of embryogenic clumps (EMCs) in cultures during the proliferation phase. K_L a varied between 0.11 and 1.47 h⁻¹ without biomass production being limited by oxygen availability. The EMCs grew even in hypoxic conditions, although the suppression of gaseous exchange strongly reduced biomass production. Cultures with different levels of hydrodynamic stress and SFI values (1.4·10⁻³–8.8·10⁻³ cm min⁻¹) were obtained. Proliferation rates of EMCs increased with agitation rate and the SFI. The largest number of EMCs was obtained in baffled flasks agitated at 160 rpm (K_L a of 1.47 h⁻¹, and SFI of 8.8·10⁻³ cm min⁻¹) with mild hydrodynamic stress enhancing growth. Biomass production increased with agitation and hydrodynamic stress, but only when the SFI value was below 5·10⁻³ cm min⁻¹. The greatest biomass production was obtained in smooth 100 ml flasks agitated at 160 rpm. The differentiation of embryos was favoured by the lowest K_L a (0.11 h⁻¹) and SFI (1.40·10³ cm min⁻¹) values, achieved using these flasks when agitated at 60 rpm.     

The effect of hydrodynamic stress on the growth of Xanthomonas campestris cultures in a stirred and sparged tank bioreactor

The specific growth and the xanthan production rates by the bacterium Xanthomonas campestris under different shear levels in shake flasks and in a stirred and sparged tank bioreactor have been studied. The shake flask has been used as a reference for studying the shear effects. An effectiveness factor expressed by the ratio of the observed growth rate and the growth rate without oxygen limitation or cell damage was calculated in both modes of cultures. It was observed that the effectiveness factor was strongly dependent on the operational conditions. A strong oxygen transfer limitation at low stirring rates, indicated by a 54 % decrease in the effectiveness factor was observed. In contrast, at higher stirrer speed, cell damage was caused by hydrodynamic stress in the turbulent bulk of the broth, yielding again a decrease in the effectiveness factor values for stirrer speeds higher than 500 rpm. Cell morphological changes were also observed depending on the agitation conditions, differences in morphology being evident at high shear stress.     

Gene expression analysis of a Louisiana native Chlorella vulgaris (Chlorophyta)/Leptolyngbya sp. (Cyanobacteria) co‐culture using suppression subtractive hybridization

A locally isolated co‐culture of two photosynthetic species [Chlorella vulgaris (Chlorophyta) and Leptolyngbya sp. (Cyanobacteria)] displayed enhanced growth when compared to a Chlorella monoculture; however, the biological mechanisms driving such improvement are currently not well understood. To investigate these mechanisms, this study examined the differential gene expression in the Chlorella between the co‐culture and the monoculture. Suppression subtractive hybridization was performed between mRNA from Chlorella in the co‐culture and in a monoculture, and 105 genes were identified as being putatively differentially expressed. Nine of these genes, corresponding to the key functional categories of energy, metabolism, and protein synthesis, were further examined using quantitative real‐time PCR and showed differential regulation of photosystem I and photosystem II and upregulation of stress‐response genes and a gene encoding an oil‐globule‐associated gene in the co‐culture Chlorella. This differential gene expression study of a Chlorella/cyanobacteria co‐culture will aid in the development of culture strategies capable of taking advantage of these differences for the production of biomass and bioproducts of interest. Knowledge of the underlying genetic causes of the changes in growth and productivity of the species in co‐culture provides insights on possible target genes for optimization of the culture.     

Responses of the desert green algae,Chlorella sp. to drought stress

Desert algae are important components of the desert soil crust and play an essential role in desert soil ecosystem development. Owing to their special habitat, desert algae are often exposed to harsh environments, among which drought represents the most common stress. Green algae are considered to have drought tolerance potential; however, only a few studies have investigated this. In this study, we selected the green alga Chlorella sp., which was isolated from desert soil, and studied its physiological response to polyethylene glycol (PEG) 6000-induced drought stress. The results showed that drought stress can affect the photosynthetic efficiency of Chlorella sp., reduce its water retention ability, and destroy its ultrastructure. However, Chlorella sp. can cope with drought stress through a series of physiological regulatory strategies. Protective strategies include quick recovery of photosynthetic efficiency and increased chlorophyll content. In addition, induced synthesis of soluble proteins, lipids, and extracellular polysaccharide (EPS), and accumulation of osmotic regulatory substances, such as sucrose and trehalose, also contribute to improving drought tolerance in Chlorella sp. This study provides insights into the physiological responses of Chlorella sp. to drought stress, which may be valuable for understanding the underlying drought adaptation mechanisms of desert green algae.     

Causes of shear sensitivity of the toxic dinoflagellate Protoceratium reticulatum

Dinoflagellates have proven extremely difficult to culture because they are inhibited by low‐level shear forces. Specific growth rate of the toxic dinoflagellate Protoceratium reticulatum was greatly decreased compared with static control culture by intermittent exposure to a turbulent hydrodynamic environment with a bulk average shear rate that was as low as 0.3 s^?1. Hydrodynamic forces appeared to induce the production of reactive oxygen species (ROS) within the cells and this caused peroxidation of cellular lipids and ultimately cell damage. Exposure to damaging levels of shear rate correlated with the elevated level of lipoperoxides in the cells, but ROS levels measured directly by flow cytometry did not correlate with shear induced cell damage. This was apparently because the measured level of ROS could not distinguish between the ROS that are normally generated by photosynthesis and the additional ROS produced as a consequence of hydrodynamic shear forces. Continuously subjecting the cells to a bulk average shear rate value of about 0.3 s^?1 for 24‐h caused an elevation in the levels of chlorophyll a, peridinin and dinoxanthin, as the cells apparently attempted to counter the damaging effects of shear fields by producing pigments that are potential antioxidants. In static culture, limitation of carbon dioxide produced a small but measureable increase in ROS. The addition of ascorbic acid (0.1 mM) to the culture medium resulted in a significant protective effect on lipid peroxidation, allowing cells to grow under damaging levels of shear rates. This confirmed the use of antioxidant additives as an efficient strategy to counter the damaging effects of turbulence in photobioreactors where shear sensitive dinoflagellates are cultivated. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Computational modeling of shear forces and experimental validation of endothelial cell responses in an orbital well shaker system

Vascular endothelial cells are continuously exposed to hemodynamic shear stress. Intensity and type of shear stress are highly relevant to vascular physiology and pathology. Here, we modeled shear stress distribution in a tissue culture well (R = 17.5 mm, fill volume 2 ml) under orbital translation using computational fluid dynamics with the finite element method. Free surface distribution, wall shear stress, inclination angle, drag force, and oscillatory index on the bottom surface were modeled. Obtained results predict nonuniform shear stress distribution during cycle, with higher oscillatory shear index, higher drag force values, higher circular component, and larger inclination angle of the shear stress at the periphery of the well compared with the center of the well. The oscillatory index, inclination angle, and drag force are new quantitative parameters modeled in this system, which provide a better understanding of the hydrodynamic conditions experienced and reflect the pulsatile character of blood flow in vivo. Validation experiments revealed that endothelial cells at the well periphery aligned under flow and increased Kruppel-like Factor 4 (KLF-4), cyclooxygenase-2 (COX-2) expression and endothelial nitric oxide synthase (eNOS) phosphorylation. In contrast, endothelial cells at the center of the well did not show clear directional alignment, did not induce the expression of KLF-4 and COX-2 nor increased eNOS phosphorylation. In conclusion, this improved computational modeling predicts that the orbital shaker model generates different hydrodynamic conditions at the periphery versus the center of the well eliciting divergent endothelial cell responses. The possibility of generating different hydrodynamic conditions in the same well makes this model highly attractive to study responses of distinct regions of the same endothelial monolayer to different types of shear stresses thereby better reflecting in vivo conditions.     

Correlation of O2 uptake rate and mitochondrial activity in carrot cell cultures exposed to laminar fluid shear

Measuring uncoupled oxygen uptake rate (OUR) could provide a convenient method for quantifying changes in the metabolic activity of plant cultures caused by hydrodynamic shear. Experiments on Daucus carota (carrot) cells were performed in a novel O₂-permeable Couette viscometer at varying levels of laminar shear (6 to 100 N m^–2). When the uncoupled OUR of the cells was compared with mitochondrial activity (determined by 2,3,5-triphenyl tetrazolium chloride assay), a significant correlation was observed (R=0.91 by linear regression).     

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.     

Factors Affecting the Mixotrophic Flagellate Poterioochromonas malhamensis Grazing on Chlorella Cells

Grazing behaviour between protozoa and phytoplankton exists widely in planktonic ecosystems. Poterioochromonas malhamensis is a well‐known and widespread mixotrophic flagellate, which is recognized to play an important role within marine and freshwater planktonic ecosystems and regarded as the greatest contamination threat for mass algal cultures of Chlorella. In this study, a comprehensive range of factors, including morphological characters, biochemical compositions, and specific growth rate of ten species or strains of Chlorella, were evaluated for their effect on the feeding ability of P. malhamensis, which was assessed by two parameters: the clearance rate of P. malhamensis on Chlorella spp. and the specific growth rate of P. malhamensis. The results showed that the clearance rate of P. malhamensis was negatively correlated with cell wall thickness and specific growth rate of Chlorella spp., while the specific growth rate of P. malhamensis was positively correlated with carbohydrate percentage and C/N ratio and negatively correlated with protein, lipid percentage, and nitrogen mass. In conclusion, the factors influencing feeding selectivity include not only the morphological character and chemical composition of Chlorella, but also its population dynamics. Our study provides useful insights into the key factors that affect the feeding selectivity of P. malhamensis and provides basic and constructive data to help in screening for grazing‐resistant microalgae.     

Formation of α-keto acids from amino acids using immobilized bacteria and algae

Summary Both free and immobilized cells of the algae Chlorella vulgaris and Anacystis nidulans contain aminoacid oxidase (AAO) activity which is increased by illumination with red light. Both immobilized species are photosynthetically active. By co-immobilizing Chlorella with bacterial cells (Providencia sp. PCM 1298) containing high AAO activity an increased production of keto acid (up to tenfold) is observed due to improved oxygen supply.     

Production of <Emphasis Type="Italic">Chlorella</Emphasis> biomass enriched by selenium and its use in animal nutrition: a review

Feedstuffs are routinely supplemented with various selenium sources, where organic forms of Se are more bio-available and less toxic than the inorganic forms (selenites, selenates). When the algae are exposed to environmental Se in the form of selenite, they are able as other microorganisms to incorporate the element to different levels, depending on the algae species. Technology of heterotrophic fed-batch cultivation of the microalga Chlorella enriched by organically bound Se was developed, where the cultivation proceeds in fermentors on aerated and mixed nutrient solution with urea as a nitrogen and glucose as a carbon and energy source. High volumetric productivity and high cell concentrations (about 70–100 g Chlorella dry mass l⁻¹) can be attained if nutrients and oxygen are adequately supplied. Addition of a small quantity of a new selenoprotein source-spray-dried Se-Chlorella biomass to the diet of farm animals had better effects on specific physiological and physical parameters of animals than selenite salt and was comparable with Se yeast added to the diet. This review introduces the importance of selenium for humans and animals, methods of Se determination, heterotrophic production of selenium-enriched Chlorella biomass in a fed-batch culture regime on organic carbon, and use of the biomass in animal nutrition.     

Shear stress effects on production of exopolymeric substances and biofilm characteristics during phenol biodegradation by immobilized Pseudomonas desmolyticum (NCIM2112) cells in a pulsed plate bioreactor

This article reports studies on a continuous pulsed plate bioreactor (PPBR) with the cells of Pseudomonas desmolyticum (NCIM2112) immobilized on granular activated carbon (GAC) used as a biofilm reactor for biodegradation of phenol. Almost complete removal of 200 ppm phenol could be achieved in this bioreactor. Biofilm structure and characteristics are influenced by hydrodynamic and shear conditions in bioreactors. In this article, the effect of shear stress induced by frequency of pulsation on biofilm characteristics during the startup period in the PPBR is reported. The startup time decreased with the increase in frequency of pulsation. The formation of biofilm in PPBR was found to have three phases: accumulation, compaction, and plateau. The effect of frequency on production of exoploymeric substances (EPS) such as, protein, carbohydrate, and humic substance is reported. An increase in shear stress induced by the frequency of pulsation increased the production of exopolymeric substances in the biofilm during startup of the bioreactor. Increase in shear stress caused a decrease in biofilm thickness and an increase in dry density of the biofilm. Increase in shear stress resulted in a smoother and thinner biofilm surface with more compact and dense structure.     

Nitrogen Starvation Induced Oxidative Stress in an Oil-Producing Green Alga Chlorella sorokiniana C3

Microalgal lipid is one of the most promising feedstocks for biodiesel production. Chlorella appears to be a particularly good option, and nitrogen (N) starvation is an efficient environmental pressure used to increase lipid accumulation in Chlorella cells. The effects of N starvation of an oil-producing wild microalga, Chlorella sorokiniana C3, on lipid accumulation were investigated using thin layer chromatography (TLC), confocal laser scanning microscopy (CLSM) and flow cytometry (FCM). The results showed that N starvation resulted in lipid accumulation in C. sorokiniana C3 cells, oil droplet (OD) formation and significant lipid accumulation in cells were detected after 2 d and 8 d of N starvation, respectively. During OD formation, reduced photosynthetic rate, respiration rate and photochemistry efficiency accompanied by increased damage to PSII were observed, demonstrated by chlorophyll (Chl) fluorescence, 77K fluorescence and oxygen evolution tests. In the mean time the rate of cyclic electron transportation increased correspondingly to produce more ATP for triacylglycerols (TAGs) synthesis. And 0.5 d was found to be the turning point for the early stress response and acclimation of cells to N starvation. Increased level of membrane peroxidation was also observed during OD formation, and superoxide dismutase (SOD), peroxide dismutase (POD) and catalase (CAT) enzyme activity assays suggested impaired reactive oxygen species (ROS) scavenging ability. Significant neutral lipid accumulation was also observed by artificial oxidative stress induced by H₂O₂ treatment. These results suggested coupled neutral lipid accumulation and oxidative stress during N starvation in C. sorokiniana C3.     

Modeling of lutein production by heterotrophic <Emphasis Type="Italic">Chlorella</Emphasis> in batch and fed-batch cultures

Chlorella is a promising alternative resource of lutein (xanthophyll) production as it can be cultivated heterotrophically in fermentors. In this paper, a kinetic model for lutein production by heterotrophic Chlorella pyrenoidosa was developed based on batch cultivations in 250-ml flasks and a 19-l fermentor. The model was validated by experimental data from two fed-batch cultivations performed in the same fermentor. The dynamic behavior of lutein production by C. pyrenoidosa with various concentrations of glucose and nitrogen was analyzed based on the kinetic model. Model-based analyses suggested that glucose concentrations between 5 and 24 g/l and nitrogen concentrations between 0.7 and 12 g/l during the cultivation were favorable for lutein production by heterotrophic C. pyrenoidosa. It also showed that fed-batch cultivations are more suitable for efficient production of lutein than batch ones. The results obtained in this study may contribute to commercial lutein production by heterotrophic Chlorella.     

三峡水库神农溪2014年春季浮游藻类演替成因分析

摘要：【目的】研究三峡水库神农溪库湾春季水华期间浮游藻类演替及其成因分析。【方法】2014年3–5月在神农溪库湾布置了6个断面(SN01–SN06),在神农溪汇入长江干流河口附近水域设置1个断面CJBD,对浮游藻类、相关环境因子及水动力因子进行了同步监测,据此分析了水体层化结构及水动力特性。【结果】神农溪在监测时段内共检测到浮游藻类6门38种(属);库湾浮游藻类生物量时间上差异显著(ANOVA,P<0.05)。春季浮游藻类群落结构具有明显的演替规律,3月份暴发大面积的硅藻水华（藻密度>100×105 cells/L),小环藻(Cyclotella spp.)为优势藻种;4月在SN02–SN06暴发以小球藻(Chlorella spp.)为主要优势种、衣藻(Chlamydomonas spp.)为次优势种的绿藻水华(藻密度>100×105 cells/L),5月份受水位大幅消落影响,浮游藻类生物量降低且无明显优势藻种。【结论】在具备充足的营养盐的水体中,水体层化结构与水动力特性对浮游藻类演替影响重大。三峡水库水位处于快速消落阶段时,流速成为抑制神农溪库湾藻类生长的主要因素。     

Production of anti‐inflammatory compounds in Sphaeralcea angustifolia cell suspension cultivated in stirred tank bioreactor

Sphaeralcea angustifolia is a plant used for the treatment of inflammatory processes. Scopoletin, tomentin, and sphaeralcic acid were identified as the compounds with anti‐inflammatory and immunomodulatory effects. Successful establishment of the cell culture in Erlenmeyer flasks has been reported previously. The aim of this study was to evaluate the ability of cells in suspension from S. angustifolia grown in a stirred tank bioreactor and demonstrate their capacity to produce bioactive compounds. Cells in suspension grown at 200 rpm reached a maximal cell biomass in dry weight at 19.11 g/L and produced 3.47 mg/g of sphaeralcic acid. The mixture of scopoletin and tomentin was only detected at the beginning of the culture (12.13 μg/g). Considering that the profile of dissolved oxygen during the cultures was lesser than 15%, it is possible that the low growth at 100 rpm could be due to oxygen limitations or to cell sedimentation. At 400 rpm, a negative effect on cell viability could be caused by the increase in the hydrodynamic stress, including the impeller tip, average shear rate, and Reynolds number. The sphaeralcic acid content in the cell suspension of S. angustifolia obtained in the bioreactor was two orders of magnitude greater than that reported for the culture grown in Erlenmeyer flasks.     

Symbiotic Chlorella enhances the thermal tolerance in Paramecium bursaria

Symbiotic Chlorella enhanced the tolerance to high temperature in Paramecium bursaria. We found that 50% of Chlorella-free P. bursaria died within 85 s of exposure to 41°C in a standard saline solution, while the presence of Chlorella almost doubled the survival time of P. bursaria (160 s, P<0.001). The degree of tolerance in 3-(3,4-dichlorophenyl)-1,1-dimethylurea (an inhibitor for photosynthesis) treated Chlorella-containing P. bursaria and Chlorella-containing organisms kept in the dark for 24 h was as low as in Chlorella-free organisms. The degree of tolerance to high temperature in Chlorella-free P. bursaria in solutions containing maltose, glucose, fructose or O₂, was as high as that of normal Chlorella-containing organisms. The degree of thermal tolerance in Chlorella-containing P. bursaria was not affected in the presence of these carbohydrates or oxygen.