Low Cell Density Cultures Research Articles

High cell density culture of microalgae in horizontal thin-layer algal reactor: Modeling of light attenuation and cell growth kinetics

This study delved into the light intensity effects and light attenuation modeling in high cell density culture (HCDC) of green alga Neochloris oleoabundans. The research primarily focused on how different light intensities influence cell growth in terms of cell division and cell mass, and the biochemical composition within the cells. Recognizing the need to avoid overestimating light path by excluding data point in the data zone where the light intensity was zero, we proposed and verified a novel modified Beer-Lambert model, which was superb in fitting experimental data and predicting light attenuation in both low and high cell density cultures. Taking advantage of the prediction power of the modified Beer-Lambert model, we devised an approach to maintain constant mean light intensity (Imean) and by adjusting the incident light intensity (I0). The results indicate that at an Imean of approximately 66.67 μmol/m2/s and 6 mm culture thickness, maximum volumetric and areal biomass productivities of 2.72 g/L/day and 16.32 g/m2/day, respectively, were achieved. Whereas the highest biomass concentration of 28.20 g/L was produced in 20 days at Imean 50 μmol/m2/s. Photoinhibition to cell division become evident at I0 750 μmol/m2/s and above. Using the data generated at constant Imean in the constant specific growth rate range, superb fitting to the Monod model with a R2 of 0.9910 was demonstrated, highlighting the importance of generating reliable data for the modelling of the kinetics of photoautotrophic growth of microalgae, which had considered to be challenging.

Optimization of Culture Media and Feeding Strategy for High Titer Production of an Adenoviral Vector in HEK 293 Fed-Batch Culture.

Adenoviruses are efficient and safe vectors for delivering target antigens and adenovirus-based vaccines have been used against a wide variety of pathogens, including tuberculosis and COVID-19. Cost-effective and scalable biomanufacturing processes are critical for the commercialization of adenovirus-vectored vaccines. Adenoviral vectors are commonly produced through the infection of batch cultures at low cell density cultures, mostly because infections at high cell densities result in reduced cell-specific virus productivity and does not improve volumetric productivity. In this study, we have investigated the feasibility of improving the volumetric productivity by infecting fed-batch cultures at high cell densities. Four commercial and one in-house developed serum-free media were first tested for supporting growth of HEK 293 cells and production of adenovirus type 5 (Ad5) in batch culture. Two best media were then selected for development of fed-batch culture to improve cell growth and virus productivity. A maximum viable cell density up to 16 × 106 cells/mL was achieved in shake flask fed-batch cultures using the selected media and commercial or in-house developed feeds. The volumetric virus productivity was improved by up to six folds, reaching 3.0 × 1010 total viral particles/mL in the fed-batch culture cultivated with the media and feeds developed in house and infected at a cell density of 5 × 106 cells/mL. Additional rounds of optimization of media and feed were required to maintain the improved titer when the fed-batch culture was scaled up in a bench scale (3 L) bioreactor. Overall, the results suggested that fed-batch culture is a simple and feasible process to significantly improve the volumetric productivity of Ad5 through optimization and balance of nutrients in culture media and feeds.

The Increase in the Drug Resistance of Acute Myeloid Leukemia THP-1 Cells in High-Density Cell Culture Is Associated with Inflammatory-like Activation and Anti-Apoptotic Bcl-2 Proteins.

It is known that cell culture density can modulate the drug resistance of acute myeloid leukemia (AML) cells. In this work, we studied the drug sensitivity of AML cells in high-density cell cultures (cell lines THP-1, HL-60, MV4-11, and U937). It was shown that the AML cells in high-density cell cultures in vitro were significantly more resistant to DNA-damaging drugs and recombinant ligand izTRAIL than those in low-density cell cultures. To elucidate the mechanism of the increased drug resistance of AML cells in high-density cell cultures, we studied the activation of Bcl-2, Hif-1alpha, and NF-kB proteins, as well as cytokine secretion, the inflammatory immunophenotype, and the transcriptome for THP-1 cells in the low-density and high-density cultures. The results indicated that the increase in the drug resistance of proliferating THP-1 cells in high-density cell cultures was associated with the accumulation of inflammatory cytokines in extracellular medium, and the formation of NF-kB-dependent inflammatory-like cell activation with the anti-apoptotic proteins Bcl-2 and Bcl-xl. The increased drug resistance of THP-1 cells in high-density cultures can be reduced by ABT-737, an inhibitor of Bcl-2 family proteins, and by inhibitors of NF-kB. The results suggest a mechanism for increasing the drug resistance of AML cells in the bone marrow and are of interest for developing a strategy to suppress this resistance.

Low-density cell culture enhances hepatic function through tight junction formation in HepG2 cells.

An in vitro evaluation system using cultured hepatocytes is the most useful method in preclinical research, such as drug metabolism and toxicity test. Human hepatocytes should be used in an in vitro evaluation system because the expression of drug-metabolizing enzymes varies among animal species. HepG2 cells, a liver cancer-derived cell line, are widely used as a human hepatocyte model; however, their hepatic functions are generally weak. In this study, we showed that low-density HepG2 cell culture induces hepatic function. The morphology of HepG2 cells was altered depending on the cell density at the time of seeding. Low-density cultured HepG2 cells proliferated as tightly packed colonies. The HepG2 cell colonies in low-density culture demonstrated enhanced tight junction formation. Tight junction protein gene expression levels, such as those of zonula occludens-1 (ZO-1), junctional adhesion molecule 1 (JAM), claudin, occludin, and tricellulin, increased in low-density cultured HepG2 cells. Phases I and II metabolic enzymes, phase III transporter gene expression, and CYP3A4 activity also increased in low-density cultured HepG2 cells. Occludin and tricellulin knockdown inhibited the increased hepatic function in low-density cultures. Tricellulin knockdown reduced the expression of hepatocyte nuclear factor 6 (HNF6), CCAAT/enhancer-binding protein alpha (CEBPA), and aryl hydrocarbon receptor (AHR). In addition, the expression of nuclear receptor subfamily 1 group h member 2 (NR1H2) increased in low-density cultures, canceled by occludin and tricellulin knockdown. Our results suggest that low-density HepG2 cell cultures enhance hepatic function by promoting tight junction formation and demonstrate the importance of cell density in drug evaluation using hepatocyte cell lines.

Massive expansion of human induced pluripotent stem cells resulting in efficient biobanking and functional 3D tissue analysis of genetic cardiomyopathies

Abstract Introduction Over the past decade, various protocols were established to ensure efficient differentiation of hiPSC into cardiomyocytes (CMs). A major limitation, however, remained the batch-to-batch variability of hiPSC-CM efficiency and cell number. Here, we suggest an approach in which concomitant GSK-3β inhibition and removal of cell-cell contact inhibition, resulted in a massive proliferative response of hiPSC-CMs1–3. This efficient method allows expansion and passaging of functional hiPSC-CMs, that routinely can be cryopreserved and subsequently used as a stable cell source for the downstream applications, such 3D in vitro models for the disease modelling of dilated cardiomyopathy (DCM). We focussed on the deletion of arginine 14 in the PLN gene (R14del), which is associated with severe heart failure in DCM patients, associated with arrhythmias, cardiac fibrosis and premature death. Methods Subsequent expansion of hiPSC-CM cultures is generally modest (<10 fold). Here, we describe a cost-effective strategy for massive expansion (up to 250-fold) of high-purity hiPSC-CMs relying on two aspects; 1) inhibition of cell-cell contact via low-density seeding and serial passaging in culture flask-format, 2)small molecular glycogen synthase kinase-3β inhibition with CHIR99021 (CHIR). Patient-specific hiPSC-CMs harbouring a PLNR14del mutation were generated and used for EHT formation and functional follow-up. Results We observed that proliferating hiPSC-CMs, especially within the first 2 passages, can routinely be cryopreserved and subsequently further expanded or utilized in downstream applications. Moreover, using this strategy, it is possible to produce ultimately >1 billion CMs within 3–5 weeks starting with one differentiation batch of day 11 hiPSC-CMs, without the need for cell sorting or selection. Expanded hiPSC-CMs retain their capacity to mature and allows fibrin-based engineered heart tissues (EHTs) formation. Previously expanded CMs from PLNR14del patient-specific hiPSC were used to generate EHT and displayed a reduced force phenotype (0.137±0.012 mN) vs healthy control (0.229±0.030 mN) and isogenic control (0.224±0.008 mN) in previously expanded CMs. Conclusion We provpresent a novel strategy for the massive expansion of functional hiPSC-CMs with concomitant GSK-3β inhibition and low cell density culture that ultimately generates up to a 250-fold increase in hiPSC-CM numbers. Expansion healthy control hiPSC-CMs does not limit the subsequent maturation process, and moreover cells remain fully functional such as required for downstream tissue engineering approaches. Therefore, CM expansion forms a well-controlled platform for upscaling hiPSC-CM production for functional 3-dimensionale PLN cardiac disease models, large drug screenings and multiple translational/regenerative applications. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): PLN Foundation

A New Non-invasive Technique for Measuring 3D-Oxygen Gradients in Wells During Mammalian Cell Culture.

Oxygen tension plays an important role in overall cell function and fate, regulating gene expression, and cell differentiation. Although there is extensive literature available that supports the previous statement, little information is to be found about accurate O2 measurements during culture. In fact, O2 concentration at the cell layer during culture is commonly assumed to be equal to that of the incubator atmosphere. This assumption does not consider oxygen diffusion properties, cell type, cell density, media composition, time in culture nor height of the cell culture medium column. In this study, we developed a non-invasive, optical sensor foil-based technique suitable for measuring the 3D oxygen gradient that is formed during cell culture as a result of normal cell respiration. For this propose, we created a 3D printed ramp to which surface an oxygen optode sensor foil was attached. The ramps were positioned inside the culture wells of 24 well plate prior cell seeding. This set up in conjunction with the VisiSens TD camera system allows to investigate the oxygen gradient formation during culture. Cultivation was performed with three different initial cell densities of the cell line A549 that were seeded on the plate containing the ramps with the oxygen sensors. The O2 gradient obtained after 96 h of culture showed significantly lower O2 concentrations closer to the bottom of the well in high cell density cultures compared to that of lower cell density cultures. Furthermore, it was very interesting to observe that even with low cell density culture, oxygen concentration near the cell layer was lower than that of the incubator atmosphere. The obtained oxygen gradient after 96 h was used to calculate the oxygen consumption rate (OCR) of the A549 cells, and the obtained value of ~100 fmol/h/cell matches the OCR value already reported in the literature for this cell line. Moreover, we found our set up to be unique in its ability to measure oxygen gradient formation in several wells of a cell culture plate simultaneously and in a non-invasive manner.

Shear stress computation in a millimeter thin flat panel photobioreactor: Numerical design validated by experiments

Flat panels are the most spread type of photobioreactors for studying light effects on a microalgae culture. Their low thickness, usually between 1 and 3cm, aims at ensuring light homogeneity across the culture. Yet because optical density has to remain very low, studies are still limited to low cell density cultures. To alleviate this problem, even thinner photobioreactors can be designed. Nevertheless, thin flat panel reactors are very prone to induce high shear stress. This work aimed at designing a new millimeter thin panel photobioreactor to study light effects on Chlorella and Scenedesmus genera. We proposed a numerical workflow that is capable of assessing the shear stress intensity in such a reactor. The numerical predictions were validated at three different levels: 2D preliminary simulations were able to reproduce bubble commonly known behaviors; close to the nozzle, the predictions were successfully confronted to shadowgraphy experimental reference; at the mini bioreactor scale, experimental and numerical mixing were found to be close. After these throughout validations, shear stress intensity in the photobioreactor was calculated over 1000 Lagrangian tracers. The experienced shear stress was agglomerated at the population level. From the computed shear stress, it was possible to choose the minimal reactor thickness that would not hinder cell growth.

Computer time-lapse microscopy of living cells with the TSITOMIR video complex: scientific, medical, and ecological possibilities

Reliable testing of effects of various natural and synthetic substances on cells is critical in searching for effective inhibitory or stimulating agents. Such work is essential in the discovery of anticancer drugs and testing toxicity of newly synthesized compounds. We use the TSITOMIR (“World of Cells”) computer video complex developed in cooperation with the Planar Corporation (Minsk). This complex is intended for time-lapse video microscopy of living cells (mouse and human fibroblasts, cells from human hair follicle and urine, cancer cultures). We elaborated a method for counting cells in low-density cell cultures to calculate the number of cells in the same growth substrate areas during cultivation, and to build cell genealogy trees. We also developed a non-invasive technology for receiving and analysing cells from human hair follicle and urine. It was shown that the cells obtained aged after a two-week cultivation, and therefore were not prone to immortalization, i.e., suitable for use in research for regenerative medicine. The method allows to obtain cell numbers in dynamics and makes it possible to get statistically valid quantitative data by using a minimal number of culture flasks. This approach can be exploited for testing antiproliferative and apoptosis-inducing effects, as well as the action of various substances and their combinations.

Reviving the Weizmann process for commercial n-butanol production

Developing a commercial process for the biological production of n-butanol is challenging as it needs to combine high titer, yield, and productivities. Here we engineer Clostridium acetobutylicum to stably and continuously produce n-butanol on a mineral media with glucose as sole carbon source. We further design a continuous process for fermentation of high concentration glucose syrup using in situ extraction of alcohols by distillation under low pressure and high cell density cultures to increase the titer, yield, and productivity of n-butanol production to the level of 550 g/L, 0.35 g/g, and 14 g/L/hr, respectively. This process provides a mean to produce n-butanol at performance levels comparable to that of corn wet milling ethanol plants using yeast as a biocatalyst. It may hold the potential to be scaled-up at pilot and industrial levels for the commercial production of n-butanol.

Cell fate decisions of human iPSC-derived bipotential hepatoblasts depend on cell density

During embryonic development bipotential hepatoblasts differentiate into hepatocytes and cholangiocytes- the two main cell types within the liver. Cell fate decision depends on elaborate interactions between distinct signalling pathways, namely Notch, WNT, TGFβ, and Hedgehog. Several in vitro protocols have been established to differentiate human pluripotent stem cells into either hepatocyte or cholangiocyte like cells (HLC/CLC) to enable disease modelling or drug screening. During HLC differentiation we observed the occurrence of epithelial cells with a phenotype divergent from the typical hepatic polygonal shape- we refer to these as endoderm derived epithelial cells (EDECs). These cells do not express the mature hepatocyte marker ALB or the progenitor marker AFP. However they express the cholangiocyte markers SOX9, OPN, CFTR as well as HNF4α, CK18 and CK19. Interestingly, they express both E Cadherin and Vimentin, two markers that are mutually exclusive, except for cancer cells. EDECs grow spontaneously under low density cell culture conditions and their occurrence was unaffected by interfering with the above mentioned signalling pathways.

In vitro evidences of epithelial to mesenchymal transition in low cell-density cultured human fetal hepatocytes

Culturing fetal hepatocytes in high cell-density allowed stabilization of the hepatocyte phenotype up to 8 weeks, including the maintenance of liver-specific functions. On the other hand, when cultured at low cell-density, fetal hepatocytes underwent morphological modifications and acquired fibroblastic morphology. Since a switch from E-cadherin to vimentin expression accompanied these changes, we hypothesized the occurrence of epithelial-to-mesenchymal transition when fetal hepatocytes were cultured at low cell-density. Changes in gene expressionsuch as up-regulation of fibrosis-related geneswere also observed, suggesting that the low cell-density culture system promoted the acquisition of a profibrotic phenotype in cultured hepatocytes. The origin of fibrogenic cells in the liver is not well known, and the role of hepatocytes as a source of fibrogenic cells is controversial. Therefore, we hypothesized that hepatocytes undergoing epithelial-to-mesenchymal transition could have a central role in liver fibrosis as a source of fibrogenic cells.To conclude, the high cell-density culture system could be a useful model for in vitro studies requiring long-term cultures of hepatocytes, such as the development of pharmaceutical drugs and mechanisms of viral infections. The low cell-density culture system may provide additional insights into the origin of fibrogenic cells in the liver, thus contributing to the development of novel therapeutic approaches.

Inhibition of Proliferation and Epithelial Mesenchymal Transition in Retinal Pigment Epithelial Cells by Heavy Chain-Hyaluronan/Pentraxin 3

Proliferative vitreoretinopathy (PVR) is mediated by proliferation and epithelial mesenchymal transition (EMT) of retinal pigment epithelium (RPE). Because heavy chain-hyaluronic acid/pentraxin 3 (HC-HA/PTX3) purified from human amniotic membrane exerts anti-inflammatory and anti-scarring actions, we hypothesized that HC-HA/PTX3 could inhibit these PVR-related processes in vitro. In this study, we first optimized an ARPE-19 cell culture model to mimic PVR by defining cell density, growth factors, and cultivation time. Using this low cell density culture model and HA as a control, we tested effects of HC-HA/PTX3 on the cell viability (cytotoxicity), proliferation (EGF + FGF-2) and EMT (TGF-β1). Furthermore, we determined effects of HC-HA/PTX3 on cell migration (EGF + FGF-2 + TGF-β1) and collagen gel contraction (TGF-β1). We found both HA and HC-HA/PTX3 were not toxic to unstimulated RPE cells. Only HC-HA/PTX3 dose-dependently inhibited proliferation and EMT of stimulated RPE cells by down-regulating Wnt (β-catenin, LEF1) and TGF-β (Smad2/3, collagen type I, α-SMA) signaling, respectively. Additionally, HA and HC-HA/PTX3 inhibited migration but only HC-HA/PTX3 inhibited collagen gel contraction. These results suggest HC-HA/PTX3 is a non-toxic, potent inhibitor of proliferation and EMT of RPE in vitro, and HC-HA/PTX3’s ability to inhibit PVR formation warrants evaluation in an animal model.

Quantification of Filamentous Actin (F-actin) Puncta in Rat Cortical Neurons

Filamentous actin protein (F-actin) plays a major role in spinogenesis, synaptic plasticity, and synaptic stability. Changes in dendritic F-actin rich structures suggest alterations in synaptic integrity and connectivity. Here we provide a detailed protocol for culturing primary rat cortical neurons, Phalloidin staining for F-actin puncta, and subsequent quantification techniques. First, the frontal cortex of E18 rat embryos are dissociated into low-density cell culture, then the neurons grown in vitro for at least 12-14 days. Following experimental treatment, the cortical neurons are stained with AlexaFluor 488 Phalloidin (to label the dendritic F-actin puncta) and microtubule-associated protein 2 (MAP2; to validate the neuronal cells and dendritic integrity). Finally, specialized software is used to analyze and quantify randomly selected neuronal dendrites. F-actin rich structures are identified on second order dendritic branches (length range 25-75 µm) with continuous MAP2 immunofluorescence. The protocol presented here will be a useful method for investigating changes in dendritic synapse structures subsequent to experimental treatments.

Quantification of Filamentous Actin (F-actin) Puncta in Rat Cortical Neurons.

Filamentous actin protein (F-actin) plays a major role in spinogenesis, synaptic plasticity, and synaptic stability. Changes in dendritic F-actin rich structures suggest alterations in synaptic integrity and connectivity. Here we provide a detailed protocol for culturing primary rat cortical neurons, Phalloidin staining for F-actin puncta, and subsequent quantification techniques. First, the frontal cortex of E18 rat embryos are dissociated into low-density cell culture, then the neurons grown in vitro for at least 12-14 days. Following experimental treatment, the cortical neurons are stained with AlexaFluor 488 Phalloidin (to label the dendritic F-actin puncta) and microtubule-associated protein 2 (MAP2; to validate the neuronal cells and dendritic integrity). Finally, specialized software is used to analyze and quantify randomly selected neuronal dendrites. F-actin rich structures are identified on second order dendritic branches (length range 25-75 µm) with continuous MAP2 immunofluorescence. The protocol presented here will be a useful method for investigating changes in dendritic synapse structures subsequent to experimental treatments.

1,4-Dihydropyridine Derivatives: Dihydronicotinamide Analogues-Model Compounds Targeting Oxidative Stress.

Many 1,4-dihydropyridines (DHPs) possess redox properties. In this review DHPs are surveyed as protectors against oxidative stress (OS) and related disorders, considering the DHPs as specific group of potential antioxidants with bioprotective capacities. They have several peculiarities related to antioxidant activity (AOA). Several commercially available calcium antagonist, 1,4-DHP drugs, their metabolites, and calcium agonists were shown to express AOA. Synthesis, hydrogen donor properties, AOA, and methods and approaches used to reveal biological activities of various groups of 1,4-DHPs are presented. Examples of DHPs antioxidant activities and protective effects of DHPs against OS induced damage in low density lipoproteins (LDL), mitochondria, microsomes, isolated cells, and cell cultures are highlighted. Comparison of the AOA of different DHPs and other antioxidants is also given. According to the data presented, the DHPs might be considered as bellwether among synthetic compounds targeting OS and potential pharmacological model compounds targeting oxidative stress important for medicinal chemistry.

Characterization of Liver-Specific Functions of Human Fetal Hepatocytes in Culture

This study was designed to assess liver-specific functions of human fetal liver cells proposed as a potential source for hepatocyte transplantation. Fetal liver cells were isolated from livers of different gestational ages (16-22 weeks), and the functions of cell preparations were evaluated by establishing primary cultures. We observed that 20- to 22-week-gestation fetal liver cell cultures contained a predominance of cells with hepatocytic traits that did not divide in vitro but were functionally competent. Fetal hepatocytes performed liver-specific functions at levels comparable to those of their adult counterpart. Moreover, exposure to dexamethasone in combination with oncostatin M promptly induced further maturation of the cells through the acquisition of additional functions (i.e., ability to store glycogen and uptake of indocyanine green). In some cases, particularly in cultures obtained from fetuses of earlier gestational ages (16-18 weeks gestation), cells with mature hepatocytic traits proved to be sporadic, and the primary cultures were mainly populated by clusters of proliferating cells. Consequently, the values of liver-specific functions detected in these cultures were low. We observed that a low cell density culture system rapidly prompted loss of the mature hepatocytic phenotype with downregulations of all the liver-specific functions. We found that human fetal liver cells can be cryopreserved without significant loss of viability and function and evaluated up to 1 year in storage in liquid nitrogen. They might, therefore, be suitable for cell banking and allow for the transplantation of large numbers of cells, thus improving clinical outcomes. Overall, our results indicate that fetal hepatocytes could be used as a cell source for hepatocyte transplantation. Fetal liver cells have been used so far to treat end-stage liver disease. Additional studies are needed to include these cells in cell-based therapies aimed to treat liver failure and inborn errors of metabolism.

The role of the hok/sok locus in bacterial response to stressful growth conditions

The hok/sok locus is renowned for its plasmid stabilization effect via post-segregational killing of plasmid-free daughter cells. However, the function(s) of the chromosome-encoded loci, which are more abundant in pathogenic strains of a broad range of enteric bacteria, are yet to be understood. Also, the frequent occurrence of this toxin/antitoxin addiction system in multi-drug resistance plasmids suggests additional roles. In this study, the effects of the hok/sok locus on the growth of bacteria in stressful growth-limiting conditions such as high temperature and antibiotic burden were investigated using hok/sok plasmids. The results showed that the hok/sok locus prolonged the lag phase of host cell cultures, thereby enabling the cells to adapt, respond to the stress and eventually thrive in these growth-limiting conditions by increasing the growth rate at exponential phase. The hok/sok locus also enhanced the survival and growth of cells in low cell density cultures irrespective of unfavourable growth conditions, and may complement existing or defective SOS mechanism. In addition to the plasmid stabilization function, these effects would enhance the ability of pathogenic bacteria to establish infections and propagate the antibiotic resistance elements carried on these plasmids, thereby contributing to the virulence of such bacteria.

DENSITY OF SUSPENSION CULTURE OF SOMATIC EMBRYOGENIC CELLS OF LARIX LEPTOLEPIS DETERMINES THE FATE OF DEVELOPMENT: INVOLVEMENT OF PHOSPHATE UPTAKE AND METABOLISM

Cell density of suspension culture of Larix leptolepis embryogenic cells determined the fate of the development. In a low-density cell culture, somatic embryo development was promoted, but the cells continued to proliferate and formed small spherical cell aggregates in a high-density cell culture. In the present study, we compared the uptake and metabolism of 32 P-labelled inorganic phosphate (Pi) by the cells of low- (1 %) and high (10 %)-density cultures during first 30 h. Little difference was found in the uptake of 32 Pi by the cells in both the low- and high density-cultures up to 6 h, but Pi uptake in high-density culture almost ceased at 6 hr after inoculation. In contrast, Pi uptake continued linearly in the low-density culture. 32 P was distributed in small molecular organic compounds (mainly nucleotides and sugar phosphates), nucleic acids, phospholipids, and phosphoproteins along with unmetabolized Pi. The incorporation of 32 P into nucleotide and phospholipid fractions was significantly higher in the cells of low-density culture at 30 h after inoculation. No marked difference on the endogenous levels of purine and pyrimidine nucleotides was observed in both density cultures. From these results it is speculated that the de novo nucleic acids and phospholipids synthesis required in the conversion of embryonic cells into somatic embryos may be caused by sufficient amounts of Pi from the medium occurring only in cells of the low-density culture. The nucleotide levels in both cultures were similar at least up to 72 h. Therefore, sufficient levels of nucleotides for proliferation may be supplied even in cells of the high-density culture. The involvement of Pi uptake in the phenomena observed in the low- and high-density cultures is discussed.Cell density of suspension culture of Larix leptolepis embryogenic cells determined the fate of the development. In a low-density cell culture, somatic embryo development was promoted, but the cells continued to proliferate and formed small spherical cell aggregates in a high-density cell culture. In the present study, we compared the uptake and metabolism of 32 P-labelled inorganic phosphate (Pi) by the cells of low- (1 %) and high (10 %)-density cultures during first 30 h. Little difference was found in the uptake of 32 Pi by the cells in both the low- and high density-cultures up to 6 h, but Pi uptake in high-density culture almost ceased at 6 hr after inoculation. In contrast, Pi uptake continued linearly in the low-density culture. 32 P was distributed in small molecular organic compounds (mainly nucleotides and sugar phosphates), nucleic acids, phospholipids, and phosphoproteins along with unmetabolized Pi. The incorporation of 32 P into nucleotide and phospholipid fractions was significantly higher in the cells of low-density culture at 30 h after inoculation. No marked difference on the endogenous levels of purine and pyrimidine nucleotides was observed in both density cultures. From these results it is speculated that the de novo nucleic acids and phospholipids synthesis required in the conversion of embryonic cells into somatic embryos may be caused by sufficient amounts of Pi from the medium occurring only in cells of the low-density culture. The nucleotide levels in both cultures were similar at least up to 72 h. Therefore, sufficient levels of nucleotides for proliferation may be supplied even in cells of the high-density culture. The involvement of Pi uptake in the phenomena observed in the low- and high-density cultures is discussed.

Multiple Regulatory Mechanisms Control the Expression of the Geobacillus stearothermophilus Gene for Extracellular Xylanase

Geobacillus stearothermophilus T-6 produces a single extracellular xylanase (Xyn10A) capable of producing short, decorated xylo-oligosaccharides from the naturally branched polysaccharide, xylan. Gel retardation assays indicated that the master negative regulator, XylR, binds specifically to xylR operators in the promoters of xylose and xylan-utilization genes. This binding is efficiently prevented in vitro by xylose, the most likely molecular inducer. Expression of the extracellular xylanase is repressed in medium containing either glucose or casamino acids, suggesting that carbon catabolite repression plays a role in regulating xynA. The global transcriptional regulator CodY was shown to bind specifically to the xynA promoter region in vitro, suggesting that CodY is a repressor of xynA. The xynA gene is located next to an uncharacterized gene, xynX, that has similarity to the NIF3 (Ngg1p interacting factor 3)-like protein family. XynX binds specifically to a 72-bp fragment in the promoter region of xynA, and the expression of xynA in a xynX null mutant appeared to be higher, indicating that XynX regulates xynA. The specific activity of the extracellular xylanase increases over 50-fold during early exponential growth, suggesting cell density regulation (quorum sensing). Addition of conditioned medium to fresh and low cell density cultures resulted in high expression of xynA, indicating that a diffusible extracellular xynA density factor is present in the medium. The xynA density factor is heat-stable, sensitive to proteases, and was partially purified using reverse phase liquid chromatography. Taken together, these results suggest that xynA is regulated by quorum-sensing at low cell densities.

The impacts of replacing air bubbles with microspheres for the clarification of algae from low cell-density culture

Dissolved Air Flotation (DAF) is a well-known coagulation–flotation system applied at large scale for microalgae harvesting. Compared to conventional harvesting technologies DAF allows high cell recovery at lower energy demand. By replacing microbubbles with microspheres, the innovative Ballasted Dissolved Air Flotation (BDAF) technique has been reported to achieve the same algae cell removal efficiency, while saving up to 80% of the energy required for the conventional DAF unit. Using three different algae cultures (Scenedesmus obliquus, Chlorella vulgaris and Arthrospira maxima), the present work investigated the practical, economic and environmental advantages of the BDAF system compared to the DAF system. 99% cells separation was achieved with both systems, nevertheless, the BDAF technology allowed up to 95% coagulant reduction depending on the algae species and the pH conditions adopted. In terms of floc structure and strength, the inclusion of microspheres in the algae floc generated a looser aggregate, showing a more compact structure within single cell alga, than large and filamentous cells. Overall, BDAF appeared to be a more reliable and sustainable harvesting system than DAF, as it allowed equal cells recovery reducing energy inputs, coagulant demand and carbon emissions.