Cultivation of Tetrahymena thermophila in a 1.5-m3 airlift bioreactor

A large-scale cultivation system for the mass cell production and extraction of the protozoon Tetrahymena thermophila has been developed on the basis of a low-cost complex nutrient medium. Cell growth and the production of extracellular proteases were investigated using a 15-l stirred-tank reactor and 13-l and 1500-l airlift reactors. Processes using defined and complex medium formulations were compared. After cell mass production by 1200 l cell suspension in the large airlift bioreactor, two different extraction methods, based on the use of an extraction decanter and a sedimentation procedure, were compared and followed by cell lyophilization. Cell sedimentation was shown to be the more efficient extraction method as it enabled cell retention/separation while preserving the cell structure. Maximum cell growth was achieved in the stirred-tank bioreactor, supporting the hypothesis that higher shear forces reduce the particle size of the medium, which is responsible for an optimized nutrient supply. The highest glucose uptake rates were found in defined medium lacking the nutrient particles that are present in complex medium formulations. The cell-specific proteolytic activity in culture supernatants of airlift bioreactors using complex medium conditions was higher than that of a culture broth with cells grown under defined medium formulations. Received: 24 September 1998 / Received revision: 23 November 1998 / Accepted: 29 November 1998     

Correlation of thrombosis growth rate to pathological wall shear rate during platelet accumulation

Local hemodynamics may strongly influence atherothrombosis, which can lead to acute myocardial infarction and stroke. The relationship between hemodynamics and thrombosis during platelet accumulation was studied through an in vitro flow system consisting of a stenosis. Specifically, wall shear rates (WSR) ranging from 0 to 100,000 s^?1 were ascertained through computations and compared with thrombus growth rates found by image analysis for over 5,000 individual observation points per experiment. A positive correlation (P < 0.0001) was found between thrombus accumulation rates and WSR up to 6,000 s^?1, with a decrease in growth rates at WSR >6,000 s^?1 (P < 0.0001). Furthermore, growth rates at pathological shear rates were found to be two to four times greater than for physiological arterial shear rates below 400 s^?1. Platelets did not accumulate for the first minute of perfusion. The initial lag time, before discernible thrombus growth could be found, diminished with shear (P < 0.0001). These studies show the quantitative increase in thrombus growth rates with very high shear rates in stenoses onto a collagen substrate. Biotechnol. Bioeng. 2012; 109: 2642–2650. © 2012 Wiley Periodicals, Inc.     

Adhesion of the marine fouling diatom amphora coffeaeformis to non‐solid gel surfaces

Diatom adhesion to different gel surfaces was tested under different shear conditions, using the fouling marine diatom Amphora coffeaeformis as test organism. Four polymers were selected to obtain a test matrix containing gels with different surface charge as well as different surface energies, viz. agarose, alginate, chitosan and chemically modified polyvinylalcohol (PVA‐SbQ). Three experimental systems were applied to obtain different shear rates. Experimental system 1 consisted of gels cast in a cell culturing well plate for comparing initial adhesion as well as long term biofilm development in the absence of shear. In experimental system 2, microscope slide based test surfaces were tested in aquaria under low shear conditions. A rotating annular biofilm reactor was used to obtain high and controlled shear rates. At high shear rates A. coffeaeformis cells adhered better to the charged polymer gels (alginate and chitosan) than to the low charged polymer gels (agarose and PVA‐SbQ). In the system where shear was absent A. coffeaeformis cells developed a biofilm on agarose equivalent to the charged polymer gels, while adhesion to PVA‐SbQ remained low at all shear rates. It is concluded that non‐solid surfaces did not represent an obstacle to settling and growth of this organism. As observed for solid surfaces, low charge density led to reduced attachment, particularly at high shear.     

Effects from shear stress on morphology and growth of early stages of Norway spruce somatic embryos

The shear stress effect on directional expansion of pro embryogenic masses (PEMs) and suspensor cell development of somatic embryos of Norway spruce (Picea abies) at the proliferation stage was studied by a direct and quantitative image analysis system. The experimental system allowed for detailed observations of the effect of hydrodynamic shear stress in rotating and deforming liquid cultures of proliferating Norway spruce somatic embryos. Briefly, somatic embryos at an early development stage comprised only of clusters of meristematic cells without suspensor cells were fixed on an alginate film. The alginate film was affixed on the bottom of a flow cell and the somatic embryos were subjected to laminar flow through the chamber of the flow cell. Magnified images of the cell clusters were collected every 24 h. The image data was processed based on a normalized cross‐correlation method, capable of measuring morphological and size features of individual cell clusters in both temporal and spatial domains. No suspensor cells developed in the cell clusters under shear stress of 140 s^?1 for the duration of the experiments. Cell clusters in the control cultured in stationary liquid conditions developed suspensor cells after 5–9 days in culture. Furthermore, the radial growth of meristematic cell clusters was inhibited by shear rates of 86 and 140 s^?1, corresponding to shear stress of 0.086 and 0.14 N/m², compared to growth under stationary conditions. The shear rate showed a significant negative correlation to growth rate. Control group showed no preference for direction during growth under static conditions. Biotechnol. Bioeng. 2010; 105: 588–599. © 2009 Wiley Periodicals, Inc.     

MECHANISMS OF FLUID SHEAR‐INDUCED INHIBITION OF POPULATION GROWTH IN A RED‐TIDE DINOFLAGELLATE1

Net population growth of some dinoflagellates is inhibited by fluid shear at shear stresses comparable with those generated during oceanic turbulence. Decreased net growth may occur through lowered cell division, increased mortality, or both. The dominant mechanism under various flow conditions was determined for the red‐tide dinoflagellate Lingulodinium polyedrum (Stein) Dodge. Cell division and mortality were determined by direct observation of isolated cells in 0.5‐mL cultures that were shaken to generate unquantified fluid shear. Larger volume cultures were exposed to quantified laminar shear in Couette‐flow chambers (0.004–0.019 N·m ^{? 2} shear stress) and to unquantified flow in shaken flasks. In these larger cultures, cell division frequency was calculated from flow cytometric measurements of DNA·cell^?1. The mechanism by which shear inhibits net growth of L. polyedrum depends on shear stress level and growth conditions. Observations on the isolated cells showed that shaking inhibited growth by lowering cell division without increased mortality. Similar results were found for early exponential‐phase cultures exposed to the lowest experimental shear stress in Couette‐flow chambers. However, mortality occurred when a late exponential‐phase culture was exposed to the same low shear stress and was inferred to occur in cultures exposed to higher shear stresses. Elevated mortality in those treatments was confirmed using behavioral, morphological, and physiological assays. The results predict that cell division in L. polyedrum populations will be inhibited by levels of oceanic turbulence common for near‐surface waters. Shear‐induced mortality is not expected unless shear‐stress levels are unusually high or when cellular condition resembles late exponential/stationary phase cultures.     

Enhanced chitosan production and modeling hyphal growth of Mucor rouxii interpreting the dependence of chitosan yields on processing and cultivation time

Chitosan is a major structural component of fungal cell walls and has diverse medical and other applications. However, cost‐effective culture and extraction methods for fungi need to be developed. Therefore, Mucor rouxii was grown on YPG‐media in both submerged batch and semi‐continuous cultures. Chitosan was extracted from the mycelia to explore strategies to enhance yields and production rates. As observed in earlier studies, M. rouxii is able to adapt to shear stress when cultured semi‐continuously. Modeling the hyphal growth of batch experiments shows that the mycelia were ruptured by shear forces within a short cultivation time shown by a decreased hyphal length. However, an increasing chitosan content was observed with an increasing cultivation period in semi‐continuous cultures, which is an indication for the adaption to shear stress. Semi‐continuous culture resulted in the highest contents of extractable chitosan. The results and models of hyphal growth, including tip extension and branching, suggest that repeated batch cultures may be optimal for chitosan production.     

Ferric and cupric reductase activities in the green alga Chlamydomonas reinhardtii: experiments using iron-limited chemostats

Cells of the green alga Chlamydomonas reinhardtii Dangeard were grown in Fe-limited chemostat culture over a range of growth rates (0.15–1.5 d⁻¹). Greater cell densities and culture chlorophyll levels were achieved using an excess of chelator [ethylenediamine di-(o-hydroxyphenylacetic acid)] relative to FeCl₃ (80:1), compared to growth using a 1:1 chelator:FeCl₃ ratio. The C. reinhardtii cells reduced extracellular ferric chelates, and ferric chelate reductase activity increased with increasing Fe-limited growth rates. However Fe-sufficient cells exhibited a low rate of ferric chelate reductase activity, similar to severely Fe-limited cells. Iron-limited cells were capable of reducing a wide variety of ferric chelates, representing a wide range of stability constants, at similar rates, suggesting that the stability constants of ferric complexes are not important determinants of ferric reducing activity. Cupric reductase activity also increased with increasing Fe-limited growth rates, and Cu(II) was preferentially reduced compared to Fe(III). These results suggest that both reductase activities may represent the same plasma-membrane enzyme. The rate of cupric reduction was a function of the free [Cu²⁺], not the total [Cu(II)], suggesting that free Cu²⁺ is the actual substrate for cupric reductase activity. Received: 8 July 1998 / Accepted: 5 August 1998     

Influence of temperature and pH on production of two bacteriocins by Leuconostoc mesenteroides subsp. mesenteroides FR52 during batch fermentation

The influence of temperature and pH on growth of Leuconostoc mesenteroides subsp. mesenteroides FR52 and production of its two bacteriocins, mesenterocin 52A and mesenterocin 52B, was studied during batch fermentation. Temperature and pH had a strong influence on the production of the two bacteriocins which was stimulated by slow growth rates. The optimal temperature was 20 °C for production of mesenterocin 52A and 25 °C for mesenterocin 52B. Optimal pH values were 5.5 and 5.0 for production of mesenterocin 52A and mesenterocin 52B respectively. Thus, by changing the culture conditions, production of one bacteriocin can be favoured in relation to the other. The relationship between growth and specific production rates of the two bacteriocins, as a function of the culture conditions, showed different kinetics of production and the presence of several peaks in the specific production rates during growth. Received: 13 February 1998 / Received revision: 27 May 1998 / Accepted: 1 June 1998     

Continuous production of acetone and butanol with shear-activated Clostridium acetobutylicum

Summary When using shear activation of Clostridium acetobutylicum by pumping the cells through capillaries, the cell growth, glucose consumption and product formation rates are considerably increased. Shear-activated continuous cell culture can be used as an inoculum with a welldefined fermentation activity for batch cultures. Different runs of such batch cultivation yield well-reproducible results which could not be obtained from inocula of other cultures or even of heat-shocked spores. The cells can attain a growth rate higher than 1.6 h^-1.The shear-activated continous culture growth is affected already at a butanol concentration lower than 1.6 g/l^-1.     

Dinophysis blooms in the deep euphotic zone of the Baltic Sea: do they grow in the dark?

In situ growth rates of the toxin-producing dinoflagellate Dinophysis norvegica collected in the central Baltic Sea were estimated during the summers of 1998 and 1999. Flow cytometric measurements of the DNA cell cycle of D. norvegica yielded specific growth rates (μ) ranging between 0.1 and 0.4 per day, with the highest growth rates in stratified populations situated at 15–20 m depth. Carbon uptake rates, measured using ¹⁴C incubations followed by single-cell isolation, at irradiances corresponding to depths of maximum cell abundance were sufficient to sustain growth rates of 0.1–0.2 per day. The reason for D. norvegica accumulation in the thermocline, commonly at 15–20 m depth, is thus enigmatic. Comparison of depth distributions of cells with nutrient profiles suggests that one reason could be to sequester nutrients. Measurements of single-cell nutrient status of D. norvegica, using nuclear microanalysis, revealed severe deficiency of both nitrogen and phosphorus as compared to the Redfield ratio.It is also possible that suitable prey or substrate for mixotrophic feeding is accumulating in the thermocline. The fraction of cells containing digestive vacuoles ranged from 2 to 22% in the studied populations. Infection by the parasitic dinoflagellate Amoebophrya sp. was observed in D. norvegica in all samples analysed. The frequency of infected cells ranged from 1 to 3% of the population as diel averages, ranging from 0.2 to 6% between individual samples. No temporal trends in infection frequency were detected. Estimated loss rates based on observed infection frequencies were 0.5–2% of the D. norvegica population daily, suggesting that these parasites were not a major loss factor for D. norvegica during the periods of study.     

Rheological,Mass Transfer,and Mixing Characterization of Cellulase-Producing Trichoderma reesei Suspensions

Steady and dynamic shear measurements are utilized to characterize the rheological behavior of Trichoderma reesei RUT-C30 fungal suspensions during batch growth on xylose (soluble substrate) or cellulose (particulate solid substrate) at three different fermentor impeller speeds (250, 400, and 550 rpm). Biomass concentrations versus time were unimodal on xylose and bimodal on cellulose. This behavior is consistent with relatively rapid, early growth on easily metabolized growth medium components (yeast extract), followed by a second, slower growth phase due to hydrolysis of recalcitrant cellulose by increasing cellulase concentrations. Critical dissolved oxygen (DO) concentration for T. reesei growth on cellulose was found to be 0.073 mmol/L. The DO was kept above this level by supplementing the air feed with pure oxygen, implying that mass transfer limitations were not the cause of bimodal cell growth. Steady shear rheological data showed shear thinning behavior and a yield stress for all broth samples regardless of substrate. Casson and Herschel−Bulkley constitutive equations fit steady shear data well. Dynamic shear measurements on broth suspensions indicated “gel-like” behavior at low strains, with microstructural breakdown at larger displacements. Time variations of the Casson model parameters (yield stress and Casson viscosity) and dynamic moduli (elastic and viscous modulus) followed both cell mass and morphology: a single maximum in all rheological variables resulted when cells were grown on xylose or on cellulose at impeller speeds of 400 or 550 rpm, and dual maxima were observed for cellulose-grown cells at 250 rpm.     

Differential activation of ErbB receptors in the rat olfactory mucosa by transforming growth factor-α and epidermal growth factor in vivo

Transforming growth factor-α (TGF-α) and epidermal growth factor (EGF) are members of the EGF family of growth factors. They have a common receptor, the EGF receptor. This belongs to the tyrosine kinase group of receptors called the ErbB receptor family. Other members are ErbB-2, ErbB-3, and ErbB-4. Binding of either ligand to the receptor elicits an increase in tyrosine kinase activity, resulting in the autophosphorylation of the receptor followed by a phosphorylation cascade of other tyrosine kinase substrates including mitogen-activated protein kinase (MAPK). TGF-α and EGF have been shown to stimulate cell division in the olfactory epithelium in vitro and may regulate cell division in vivo. To investigate whether exogenous TGF-α or EGF has a functional effect on the olfactory mucosa in vivo, 12.5–50 μg of each growth factor was administered to rats via the carotid artery. After 2 min, olfactory mucosa and liver samples were collected, homogenized, and immunoprecipitated with antibodies to the ErbB receptors. The immunoprecipitates were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western immunoblotting. Using phosphotyrosine antibody, the receptors were probed for phosphorylation. Activation of MAPK was also investigated using MAPK antibody. Exogenous TGF-α activated EGFR, ErbB-2 and MAPK, whereas EGF activated only the EGFR. TGF-α was a more potent activator of EGFR than EGF. Neither ligand had an effect on ErbB-3 and ErbB-4 receptors. These effects were absent in the control animals which received the same solution without the growth factor. These results are consistent with the notion that binding of TGF-α to EGFR may play a role in olfactory cell division in vivo. © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 199–210, 1998     

ENVIRONMENTAL FACTORS CONTROLLING ALEXANDRIUM TAMARENSE (DINOPHYCEAE) GROWTH RATE DURING A RED TIDE EVENT IN THE ST. LAWRENCE ESTUARY (CANADA)1

The dinoflagellate Alexandrium tamarense (Lebour) Balech 1985 is responsible for recurrent outbreaks of paralytic shellfish poisoning in the St. Lawrence Estuary. In July 1998, an A. tamarense red tide developed in the estuary with maximum cell concentrations reaching 2.3 × 10⁶ cells·L^?1 in brackish surface waters. To estimate the growth rate of these cells, surface water samples from different locations and days during the bloom were incubated for 5 to 9 days under in situ temperature and light conditions. Growth rates varied both spatially and temporally between 0 and 0.55 day^?1, reaching the maximum growth rate reported for this species in culture. High growth rates were measured even during the peak of the red tide, suggesting that the extremely high cell concentrations observed did not solely result from aggregation or physical concentration but also involved active cellular growth. Alexandrium tamarense cells were found over a large range of salinity (20.8–29.5 psu), but high densities and significant growth were only measured when salinity was lower than 24.5 psu. Under these conditions, the number of divisions achieved by A. tamarense was proportional to the amount of nitrate available at the beginning of the incubations, whereas variations in growth rate were apparently controlled by the availability of phosphate. We hypothesize that the ability of A. tamarense to perform vertical migrations and acquire nitrate at night pushes this species toward phosphate limitation in the St. Lawrence Estuary.     

Growth and abundance of Fundulus heteroclitus and Menidia menidia in estuaries of Waquoit Bay, Massachusetts exposed to different rates of nitrogen loading

To assess the effect of land-derived nitrogen loads on fish populations, we estimated the abundance and growth rate of Fundulus heteroclitus and Menidia menidia, the most common fish species found in estuaries of Waquoit Bay, from June through September in 1996, 1997 and 1998 in six estuaries subject to different rates of nitrogen loading. Abundance of F. heteroclitus was greater in estuaries with greater nitrogen loading rates, but growth rates of F. heteroclitus obtained from growth rings on scales and otoliths did not vary among estuaries. Abundance and growth rates of M. menidia did not change with increased nitrogen loading rates. Increased nitrogen load may increase abundance of some fish species within an assemblage but not others. Such bottom up controls are effective at producer trophic levels, but appear to be less pervasive with secondary consumers. Higher trophic levels, therefore, may only be loosely coupled to nutrient supply rates.     

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.     

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     

Morphology,cell growth,and polysaccharide production of <Emphasis Type="Italic">Nostoc flagelliforme</Emphasis> in liquid suspension culture at different agitation rates

Noscoc flagelliforme is a terrestrial macroscopic cyanobacterium with high economic value. Free-living cells that were separated from a natural colony of N. flagelliforme were cultivated in a 20-L photobioreactor for 16 days at five agitation rates with impeller tip speeds at 0.3, 0., 0.8, 1.0, and 1.5 m·s⁻¹. With different impeller tip speeds there were significant differences in the cell growth and polysaccharide production, and different types of cell colonies appeared because of different shear forces caused by agitation. At harvest time, cell concentrations with tip speeds of 0.8 and 1.0 m·s⁻¹ were clearly higher than those with the other three tip speeds, but dry cell weights with the tip speeds of 0.3, 0.5, 0.8, and 1.0 m·s⁻¹ were almost the same. The highest RPS (polysaccharide that released into liquid medium) production was obtained with the tip speeds of 0.8 and 1.0 m·s⁻¹, while the highest EPS (polysaccharide that formed capsule or slime layer) production was obtained with the tip speed of 0.5 m·s⁻¹. The tip speed of 1.5 m·s⁻¹ was harmful for both cell growth and polysaccharide production, indicating that an appropriate shear force was needed in the liquid suspension culture of N. flagelliforme.     

Evaluation of cell recycling in continuous fermentation of enzymatic hydrolysates of spruce with Saccharomyces cerevisiae and on-line monitoring of glucose and ethanol

The maximum growth rate of Saccharomyces cerevisiae ATCC 96581, adapted to fermentation of spent sulphite liquor (SSL), was 7 times higher in SSL of hardwood than the maximum growth rate of bakers' yeast. ATCC 96581 was studied in the continuous fermentation of spruce hydrolysate without and with cell recycling. Ethanol productivity by ATCC 96581 in continuous fermentation of an enzymatic hydrolysate of spruce was increased 4.6 times by employing cell recycling. On-line analysis of CO₂, glucose and ethanol (using a microdialysis probe) was used to investigate the effect of fermentation pH on cell growth and ethanol production, and to set the dilution rate. Cell growth in the spruce hydrolysates was strongly influenced by fermentation pH. The fermentation was operated in continuous mode for 210 h and a theoretical ethanol yield on fermentable sugars was obtained. Received: 25 May 1998 / Received revision: 11 August 1998 / Accepted: 12 August 1998