Maximum Cell Density Research Articles

Quantifying the Effects of Ethanol and Temperature on the Fitness Advantage of Predominant Saccharomyces cerevisiae Strains Occurring in Spontaneous Wine Fermentations.

Different Saccharomyces cerevisiae strains are simultaneously or in succession involved in spontaneous wine fermentations. In general, few strains occur at percentages higher than 50% of the total yeast isolates (predominant strains), while a variable number of other strains are present at percentages much lower (secondary strains). Since S. cerevisiae strains participating in alcoholic fermentations may differently affect the chemical and sensory qualities of resulting wines, it is of great importance to assess whether the predominant strains possess a “dominant character.” Therefore, the aim of this study was to investigate whether the predominance of some S. cerevisiae strains results from a better adaptation capability (fitness advantage) to the main stress factors of oenological interest: ethanol and temperature. Predominant and secondary S. cerevisiae strains from different wineries were used to evaluate the individual effect of increasing ethanol concentrations (0-3-5 and 7% v/v) as well as the combined effects of different ethanol concentrations (0-3-5 and 7% v/v) at different temperature (25–30 and 35°C) on yeast growth. For all the assays, the lag phase period, the maximum specific growth rate (μmax) and the maximum cell densities were estimated. In addition, the fitness advantage between the predominant and secondary strains was calculated. The findings pointed out that all the predominant strains showed significantly higher μmax and/or lower lag phase values at all tested conditions. Hence, S. cerevisiae strains that occur at higher percentages in spontaneous alcoholic fermentations are more competitive, possibly because of their higher capability to fit the progressively changing environmental conditions in terms of ethanol concentrations and temperature.

Scalable culture of human induced pluripotent cells on microcarriers under xeno‐free conditions using single‐use vertical‐wheel™ bioreactors

AbstractBACKGROUNDHuman induced pluripotent stem cells (hiPSC) are expected to become powerful tools for disease modelling, for the discovery and testing of new drugs and, ultimately, for regenerative therapies. The success of these applications depends on the development of scalable bioprocesses capable of generating large numbers of hiPSC and derivatives.RESULTSIn this work, the novel vertical‐wheel single‐use bioreactors were used for the first time for the expansion of hiPSC under xeno‐free conditions. Cultures were performed on microcarriers in two different scales of vessels (100 and 500 mL with 80 and 300 mL working volumes, respectively), leading to maximum cell densities up to 1.21 ± 0.02 × 106 cells mL−1 and volumetric productivities of 2.01 ± 0.04 × 105 cells mL−1 day−1. The pluripotency as well as a normal karyotype were maintained after cell expansion. Consistency of the processes was confirmed with a different hiPSC line, which is an important aspect for a personalized medicine approach.CONCLUSIONThe results here described demonstrate the feasibility of scalable production of hiPSC in a microcarrier‐based system using vertical‐wheel bioreactors. The protocols developed in this study provide a Good Manufacturing Practices (GMP)‐compliant system for hiPSC manufacturing which may be an important step towards the successful implementation of hiPSC‐based products. © 2018 Society of Chemical Industry

Insight into Energy Conservation via Alternative Carbon Monoxide Metabolism in Carboxydothermus pertinax Revealed by Comparative Genome Analysis.

Carboxydothermus species are some of the most studied thermophilic carboxydotrophs. Their varied carboxydotrophic growth properties suggest distinct strategies for energy conservation via carbon monoxide (CO) metabolism. In this study, we used comparative genome analysis of the genus Carboxydothermus to show variations in the CO dehydrogenase-energy-converting hydrogenase gene cluster, which is responsible for CO metabolism with H2 production (hydrogenogenic CO metabolism). Indeed, the ability or inability to produce H2 with CO oxidation is explained by the presence or absence of this gene cluster in Carboxydothermus hydrogenoformans, Carboxydothermus islandicus, and Carboxydothermus ferrireducens Interestingly, despite its hydrogenogenic CO metabolism, Carboxydothermus pertinax lacks the Ni-CO dehydrogenase catalytic subunit (CooS-I) and its transcriptional regulator-encoding genes in this gene cluster, probably due to inversion. Transcriptional analysis in C. pertinax showed that the Ni-CO dehydrogenase gene (cooS-II) and distantly encoded energy-converting-hydrogenase-related genes were remarkably upregulated with 100% CO. In addition, when thiosulfate was available as a terminal electron acceptor in 100% CO, the maximum cell density and maximum specific growth rate of C. pertinax were 3.1-fold and 1.5-fold higher, respectively, than when thiosulfate was absent. The amount of H2 produced was only 62% of the amount of CO consumed, less than expected according to hydrogenogenic CO oxidation (CO + H2O → CO2 + H2). Accordingly, C. pertinax would couple CO oxidation by Ni-CO dehydrogenase II with simultaneous reduction of not only H2O but also thiosulfate when grown in 100% CO.IMPORTANCE Anaerobic hydrogenogenic carboxydotrophs are thought to fill a vital niche by scavenging potentially toxic CO and producing H2 as an available energy source for thermophilic microbes. This hydrogenogenic carboxydotrophy relies on a Ni-CO dehydrogenase-energy-converting hydrogenase gene cluster. This feature is thought to be common to these organisms. However, the hydrogenogenic carboxydotroph Carboxydothermus pertinax lacks the gene for the Ni-CO dehydrogenase catalytic subunit encoded in the gene cluster. Here, we performed a comparative genome analysis of the genus Carboxydothermus, a transcriptional analysis, and a cultivation study in 100% CO to prove the hydrogenogenic CO metabolism. Results revealed that C. pertinax could couple Ni-CO dehydrogenase II alternatively to the distal energy-converting hydrogenase. Furthermore, C. pertinax represents an example of the functioning of Ni-CO dehydrogenase that does not always correspond to its genomic context, owing to the versatility of CO metabolism and the low redox potential of CO.

Temporal variability of phytoplankton biomass and primary production off Magdalena Bay, Baja California Sur, Mexico

Sixteen monthly cruises were carried out at a station 6.5 km to the NW of the mouth of Magdalena Bay (Mexico) to study the temporal variability of phytoplankton biomass and primary production rates, and the response of phytoplankton to the prevailing hydrographic characteristics. During each cruise, temperature and salinity were measured in the water column, and discrete samples were collected to measure inorganic nutrient concentrations (nitrate, phosphate, and silicate) and chlorophyll a content (as a proxy for phytoplankton biomass); in addition, in situ incubations were performed to estimate primary production (PP) rates using the 14C technique. During spring and early summer (March–July), the water column at the study site was cold (14.4 ºC at 80 m depth and 17.7 ºC at the surface) and well mixed, with high nutrient concentrations (nitrate = 6.39 μM, phosphate = 0.91 μM, and silicate = 11.87 μM) at the surface, typical of intense upwelling systems. On the contrary, from August to February the water column was stratified (23.5 ± 3.2 ºC above the thermocline and 17.0 ± 1.7 ºC at 80 m depth) and had low nutrient concentrations (nitrate = 3.90 μM, phosphate = 0.47 μM, and silicate = 10.30 μM) at the surface. Phytoplankton abundance, chlorophyll a concentration, and PP rates in the euphotic zone were higher during the upwelling season, with maximum cell density of 1.7 × 106 cells·L–1 (nanoplankton fraction), integrated chlorophyll a content of 231 mg·m–2, and integrated PP rates of 553 mg C·m–2·h–1). Abundance of cells <20 μm, as determined by flow cytometry, revealed the importance of the nanoplankton and picoplankton fractions in this region. This study showed the seasonal variability of phytoplankton communities (biomass and productivity) in waters off Magdalena Bay and the coupling of phytoplankton to environmental variability. Seasonal variability was similar to that observed for other typical coastal upwelling regions, that is, higher phytoplankton abundances and PP rates in spring and summer and lower values in autumn and winter.
 Key words: biomass, primary productivity, hydrography, coastal zone, Magdalena Bay.

FOTOBIORREACTOR TRONCO-CÓNICO Y ERA DE SECADO COMO SISTEMA EFICIENTE PARA EL CULTIVO Y RECUPERACIÓN DE LA BIOMASA SECA DE LA MICROALGA VERDE Coelastrum sp.

Microalgae are photosynthetic microorganisms that are studied due to the variety of metabolites that they produce. However, if cultivated in photobioreactors, these devices should be easy to scale up. Also, harvesting and drying the microalgal biomass should be economically viable at industrial scales. Based on this point of view, in this work a 1.5 L cylindrical-conical photobioreactor was designed to cultivate and harvest the microalga Coelastrum sp. under nitrogen limitation. Maximum cell density was 23.47 × 106 cells mL-1. Biomass and lipid productivities after 5 days of nitrogen limitation were 266.24 mg L-1 d-1 and 137.51 mg L-1 d-1 respectively. A lipid content of 45.6% (w/w dry biomass) was attained. A sludge drying bed was also designed to dry the microalgal biomass. The retention filter used in the sludge drying bed allowed a cell retention efficiency of 86.8% and the final water content of the dried biomass was 7.82 % ± 1.99 which allowed the lipid extraction without further drying. The lipid profile after transesterification of triacylglycerides was [C14:0 (3.04%), C16:0 (35.52%), C18:0 (42.50%), C18:1 (14.30%), C18:2 (4.63%)] which indicates that the lipid extract is more suitable for green diesel than biodiesel production.

The effects of syringaldehyde and vanillin on butyric acid production by fermentation using Clostridium tyrobutyricum

Butyric acid is a valuable chemical that has various applications in the chemical, pharmaceutical, food, and biofuel industries. Its bio-fermentation from lignocellulosic materials may be affected by some unwanted substrates that are generated during biomass pretreatment processes. In the present study, the potential inhibitory effects of two phenolic aldehydes (syringaldehyde and vanillin) on butyric acid fermentation by Clostridium tyrobutyricum were evaluated. The toxicity of syringaldehyde and vanillin on cell growth, xylose consumption, and butyric acid production was dosage-dependent. The butyric acid productivity decreased significantly with increasing concentrations of syringaldehyde and vanillin. C. tyrobutyricum demonstrated a broad tolerance to both syringaldehyde and vanillin and only moderate reductions in the maximum cell density were observed with up to 2.4 g L-1 of syringaldehyde or vanillin in the medium. Both syringaldehyde and vanillin were assimilated by C. tyrobutyricum, and the metabolite products from vanillin caused considerable inhibition of the fermentation.

Diel Variations in Cell Abundance and Trophic Transfer of Diarrheic Toxins during a Massive Dinophysis Bloom in Southern Brazil.

Dinophysis spp. are a major source of diarrheic toxins to marine food webs, especially during blooms. This study documented the occurrence, in late May 2016, of a massive toxic bloom of the Dinophysis acuminata complex along the southern coast of Brazil, associated with an episode of marked salinity stratification. The study tracked the daily vertical distribution of Dinophysis spp. cells and their ciliate prey, Mesodinium cf. rubrum, and quantified the amount of lipophilic toxins present in seston and accumulated by various marine organisms in the food web. The abundance of the D. acuminata complex reached 43 × 104 cells·L−1 at 1.0 m depth at the peak of the bloom. Maximum cell densities of cryptophyceans and M. cf. rubrum (>500 × 104 and 18 × 104 cell·L−1, respectively) were recorded on the first day of sampling, one week before the peak in abundance of the D. acuminata complex. The diarrheic toxin okadaic acid (OA) was the only toxin detected during the bloom, attaining unprecedented, high concentrations of up to 829 µg·L−1 in seston, and 143 ± 93 pg·cell−1 in individually picked cells of the D. acuminata complex. Suspension-feeders such as the mussel, Perna perna, and barnacle, Megabalanus tintinnabulum, accumulated maximum OA levels (up to 578.4 and 21.9 µg total OA·Kg−1, respectively) during early bloom stages, whereas predators and detritivores such as Caprellidae amphipods (154.6 µg·Kg−1), Stramonita haemastoma gastropods (111.6 µg·Kg−1), Pilumnus spinosissimus crabs (33.4 µg·Kg−1) and a commercially important species of shrimp, Xiphopenaeus kroyeri (7.2 µg·Kg−1), only incorporated OA from mid- to late bloom stages. Conjugated forms of OA were dominant (>70%) in most organisms, except in blenny fish, Hypleurochilus fissicornis, and polychaetes, Pseudonereis palpata (up to 59.3 and 164.6 µg total OA·Kg−1, respectively), which contained mostly free-OA throughout the bloom. Although algal toxins are only regulated in bivalves during toxic blooms in most countries, including Brazil, this study indicates that human seafood consumers might be exposed to moderate toxin levels from a variety of other vectors during intense toxic outbreaks.

Single Cell Study of Water Transport in PEMFCs with Electrosprayed Catalyst Layers

AbstractWater transport in proton exchange membrane fuel cells (PEMFCs) with electrosprayed catalyst layers is analyzed by means of water collection measurements. With this aim, single cells have been studied with different catalyst layers configuration: electrosprayed layer in cathode (EScat), in anode (ESan), and in both electrodes (ESboth). The electrosprayed catalyst layers show macroporous morphology and superhydrophobicity (water contact angle θ > 150°) . Initial polarization curves, taken after a few hours from cell start‐up, show above 20% higher maximum power density of EScat and ESan cells, compared with a conventional cell (with no electrosprayed layers). The performance improvement is due to lower internal resistance of the cell according to the polarization curves. Stability of the cells under the dry‐gas operation conditions used during water collection measurements, shows to be high only for EScat, whereas ESan, ESboth, and conventional cells suffer degradation and frequent membrane failure. Water collection experiments at variable temperature and current density reflect larger water flow towards the anode for EScat cell than for the other configurations. It is concluded that the high performance and stability of EScat is due to the enhanced water back diffusion from cathode to anode driven by the superhydrophobic electrosprayed catalyst layer in cathode. Favored back diffusion improves water balance in the cell, membrane humidification, decreases the internal resistance, and precludes anode drying and cathode flooding. The optimal cell configuration is obtained with an electrosprayed catalyst layer in the cathode and a standard hydrophilic catalyst layer in the anode.

The seasonal dynamics of plankton communities relative to the foraging of the southern right whale (Eubalaena australis) in northern Patagonian gulfs, Península Valdés, Argentina

This is the first exploratory study that addresses simultaneously the phytoplankton and mesozooplankton seasonal dynamics associated with some environmental factors in the northern Patagonian gulfs: Golfo Nuevo (GN) and Golfo San José GSJ), Argentina, during an annual cycle (December 2014–2015 and January 2015–2016, respectively). It also reports data on mesozooplankton composition and abundance in a sample collected while southern right whales (Eubalaena australis) were feeding at the sea surface in Bahía Pirámide (GN). The phytoplankton community was represented by 69 taxa in GN and by 83 taxa in GSJ. In GN, phytoplankton was mainly dominated by diatoms and dinoflagellates whereas in GSJ diatoms dominated the phytoplankton community throughout the year. In GN, the highest phytoplankton biomass and density values were registered during austral spring, 2015 which coincided with a Pseudo-nitzschia australis bloom. In contrast, the lowest biomass values, which were associated with elevated abundances of dinoflagellates, were observed in late spring 2014. In GSJ, the highest biomass values showed two equal peaks in austral autumn and spring 2015. The latter was associated with the maximum cell density recorded in this gulf during a Chaetoceros debilis bloom. In contrast, the lowest biomass values were recorded in winter 2015. Mesozooplankton was represented by 63 taxa in GN and by 61 taxa in GSJ. In GN, this community was mainly represented by cladocerans and copepods, whereas it was dominated by copepods in GSJ. The lowest mesozooplankton abundances were recorded in austral winter in both gulfs. In GN, the highest abundances were recorded in austral summer and spring 2015, whereas in GSJ, the maximum mesozooplankton abundances were observed during austral summer and autumn 2015. The major prey items for E. australis were the copepods Calanoides carinatus, Ctenocalanus vanus, Calanus australis, and Paracalanus parvus, zoeae of the squat lobster Munida gregaria, calyptopis and furcilia of Euphausia lucens, and fish eggs and larvae. In both gulfs, the warm seasons were mainly characterized by higher relative abundances of diatoms, while the cold seasons were mostly associated with higher abundances of dinoflagellates and Dictyochophyceae. Salinity and phaeopigments were related with copepod and appendicularian abundances, whereas surface sea water temperature seemed to modulate the seasonal distribution pattern of cladocerans and decapods.

Effect of food microstructure on growth dynamics of Listeria monocytogenes in fish-based model systems

Traditionally, predictive growth models for food pathogens are developed based on experiments in broth media, resulting in models which do not incorporate the influence of food microstructure. The use of model systems with various microstructures is a promising concept to get more insight into the influence of food microstructure on microbial dynamics. By means of minimal variation of compositional and physicochemical factors, these model systems can be used to study the isolated effect of certain microstructural aspects on microbial growth, survival and inactivation.In this study, the isolated effect on microbial growth dynamics of Listeria monocytogenes of two food microstructural aspects and one aspect influenced by food microstructure were investigated, i.e., the nature of the food matrix, the presence of fat droplets, and microorganism growth morphology, respectively. To this extent, fish-based model systems with various microstructures were used, i.e., a liquid, a second more viscous liquid system containing xanthan gum, an emulsion, an aqueous gel, and a gelled emulsion. Growth experiments were conducted at 4 and 10 °C, both using homogeneous and surface inoculation (only for the gelled systems).Results regarding the influence of the growth morphology indicated that the lag phase of planktonic cells in the liquid system was similar to the lag phase of submerged colonies in the xanthan system. The lag phase of submerged colonies in each gelled system was considerably longer than the lag phase of surface colonies on these respective systems. The maximum specific growth rate of planktonic cells in the liquid system was significantly lower than for submerged colonies in the xanthan system at 10 °C, while no significant differences were observed at 4 °C. The maximum cell density was higher for submerged colonies than for surface colonies. The nature of the food matrix only exerted an influence on the maximum specific growth rate, which was significantly higher in the viscous systems than in the gelled systems. The presence of a small amount of fat droplets improved the growth of L. monocytogenes at 4 °C, resulting in a shorter lag phase and a higher maximum specific growth rate. The obtained results could be useful in the determination of a set of suitable microstructural parameters for future predictive models that incorporate the influence of food microstructure on microbial dynamics.

Differential physiological responses of the coastal cyanobacterium Synechococcus sp. PCC7002 to elevated pCO2 at lag, exponential, and stationary growth phases

We studied the effects of expected end-of-the-century p CO2 (1000 ppm) on the photosynthetic performance of a coastal marine cyanobacterium Synechococcus sp. PCC7002 during the lag, exponential, and stationary growth phases. Elevated p CO2 significantly stimulated growth, and enhanced the maximum cell density during the stationary phase. Under ambient p CO2 conditions, the lag phase lasted for 6 days, while elevated p CO2 shortened the lag phase to two days and extended the exponential phase by four days. The elevated p CO2 increased photosynthesis levels during the lag and exponential phases, but reduced them during the stationary phase. Moreover, the elevated p CO2 reduced the saturated growth light (Ik) and increased the light utilization efficiency ( α ) during the exponential and stationary phases, and elevated the phycobilisome:chlorophyll a (Chl a ) ratio. Furthermore, the elevated p CO2 reduced the particulate organic carbon (POC):Chl a and particulate organic nitrogen (PON):Chl a ratios during the lag and stationary phases, but enhanced them during the exponential phase. Overall, Synechococcus showed differential physiological responses to elevated p CO2 during different growth phases, thus providing insight into previous studies that focused on only the exponential phase, which may have biased the results relative to the effects of elevated p CO2 in ecology or aquaculture.

Immortalization of chicken embryonic liver-derived cell line by stable expression of hMRP18S-2 for serotype 4 fowl adenovirus propagation

Inclusion body hepatitis and hydropericardium-hepatitis syndrome caused by serotype 4 fowl adenovirus (FAdV-4) have emerged in China since 2013. FAdV is usually propagated in primary chicken embryonic liver cells or embryo yolk sac. The aim of this work was to develop an immortalized CEL cell line by stable expression of human mitochondrial ribosomal protein 18S-2, named CEL-hMRP18S-2 cells, for the propagation of FAdV-4. The maximum cell density of CEL-hMRP18S-2 cells could reach 2.65 × 106 cells/ml in four-days culture. According to the mRNA levels of cell-cycle related genes in CEL-hMRP18S-2 cells tested by qRT-PCR, we speculated that the transformation of hMRP18S-2 into CEL cells caused the functional inactivation of p53 and the significant down-regulation of p15INK4b might cause the hyperphosphorylated form of Rb, releasing E2F-1 factor and enhancing the E2F-dependent transcription for cell cycle progression. It was suspected that the up-regulated c-Myc mRNA level at the initial period of immortalization might prompt transformed cells through the G0-G1 checkpoint. The normal CPE was observed in CEL-hMRP18S-2 cells infected by FAdV-4 and microcarrier suspension culture performed for FAdV-4 propagation with 9.0 lgTCID50/ml suggested that CEL-hMRP18S-2 cells could be a useful continuous cell line for isolation of wild FAdV and production of FAdV-inactivated vaccine.

A Bio-physical Assessment of Harmful Algal Bloom (HAB) Outbreak using Multi-dimensional Array Data during 1998–2017

Jeong, J.-Y.; Kang, Y.H.; Park, K.; Um, J.-H., and Son, Y.B., 2018. A bio-physical assessment of Harmful Algal Bloom (HAB) outbreak using multi-dimensional array data during 1998–2017. In: Shim, J.-S.; Chun, I., and Lim, H.S. (eds.), Proceedings from the International Coastal Symposium (ICS) 2018 (Busan, Republic of Korea). Journal of Coastal Research, Special Issue No. 85, pp. 836–840. Coconut Creek (Florida), ISSN 0749-0208.Harmful algal blooms (HABs), Cochlodinium polykrikoides, which related a devastating algal bloom, recently have caused the serious problems of fishery and aquaculture activities in the south coast of Korea (SCK). To understand the evolution of red tide and determine physical factors as a function of the bloom occurrence and disappearance, we used the multi-dimensional data (satellite and in-situ) during 1998–2017. Red tides generally have started with finishing the raining season and then increased with increasing the sea surface temperature and tidal range. To understand the general trend of red tide occurrence, we classify three different scales with cell density, area, and duration of red tides such as small, medium, and large scale red tides. Small and medium scale red tides started in the middle of August. The periods of the maximum cell density and extinction were shown in the early and end of September. SST in small and medium scale red tides was higher than in large scale red tides. Even though the tidal range was relatively small at the beginning of the outbreak, it was increased in the maximum cell density and decreased in the extinction period. Large scale red tides began in the early of August and the maximum cell density appeared at the end of August. SST was the lowest and the tidal range was the largest difference in the beginning outbreak and the lowest difference in the maximum cell density. In particular, the phenomenon of large-scale variation in the overall time series was that the SST and PAR in large scale red tides were relatively low compared to the small and medium scale red tides. Even though the tidal range was less difference before the beginning outbreak, it is analyzed that there was a growing difference in time.

Cultivation Conditions Can Cause a Shift from Mutualistic to Parasitic Behavior in the Symbiosis Between Paramecium and Its Bacterial Symbiont Caedibacter taeniospiralis.

Caedibacter taeniospiralis is an obligate bacterial symbiont living in the cytoplasm of the ciliate Paramecium tetraurelia. Different studies analyzing the effect of this symbiont on its host's growth and maximal cell density arrive at contradicting conclusions, labeling it as either a parasite or a mutualist. We address the question whether extrinsic factors such as medium and food organism are responsible for the opposing results. Thus, we performed fitness assays comparing previously applied cultivation conditions. By confirming the dependency of the parasitic and mutualistic behavior of C. taeniospiralis on the cultivation conditions of its host P. tetraurelia, we demonstrate the context-dependent impact on host fitness of this bacterium.

Study on some properties of the hybrid cell B4D10C9 producing the anti B monoclonal antibody which was agglutinated the b antigens on the surface of red blood cells

The B4D10C9 hybridoma cell line producing the monoclonal antibody which was specifically agglutinated red blood cells B group, belongs to the IgM class and kappa light chains that have been created in our previous study. This study presents some biological charateristics of B4D10C9 such as the capability of growth, anti B monoclonal antibody producing, the stability of growth and cell line and antibody secreting of this cell line. The hybridoma cells grew well in the DMEM medium added with fetal bovine serum (FBS) at the final concentration of 1% or 10%. However, the maximum density of cells reached 9,9 x 105 cells per ml after 50 hours post incubation only when it was cultured in the medium containing 10% of FBS. The maximum antibody titers in the supernatant that collected after 150 hours of culture reached 1/512. In contrast to that, the maximum density of cells only reached 3,4 x 105 cells/ml after 72 hours post incubation when was cultured in DMEM medium containing 1% FBS. The maximum antibody titer was 1/64 after 50 hours post incubation. These cells were cultured through 3 different generations and the ability of antibody production in each generation was evaluated. As a result, this cell line had continuous growth stability and antibody secretion through 3 generations. The quantity of monoclonal antibody obtained from the supenantant of the culture medium at 150 hours of incubation was about 100 micrograms per ml. Thus, the growth of the cell line B4D10C9 and its ability of secretion of anti B monoclonal antibody were better when was cultured in the DMEM medium containing 10% fetal bovine serum. Preservation, storage and recovery does not affect to the growth and antibody producing of this cell line.

Performance of La0.8Sr0.2Ga0.8Mg0.2O3-based SOFCs with atmospheric plasma sprayed La-doped CeO2 buffer layer

Ce0.6La0.4O2 (LDC) is a promising buffer layer for preventing chemical reactions between a doped-LaGaO3 electrolyte and Ni-based anode in intermediate-temperature solid oxide fuel cells (SOFCs). In the present work, first, the chemical compatibility between La0.8Sr0.2Ga0.8Mg0.2O3 (LSGM) powders and LDC powders is evaluated. Subsequently, an LDC buffer layer is prepared via atmospheric plasma spraying (APS) at different deposition temperatures using powders that have particle sizes larger than 30 μm. The effects of deposition temperature on the microstructure and ionic conductivity are investigated. Cells with an optimized APS LDC interlayer between the NiO containing anode and LSGM electrolyte are tested with humidified H2 as fuel and ambient air as the oxidation. At 750 °C, the maximum power density of cells with the APS LDC interlayer is 460 mW/cm2. In addition, a stability test of ∼400 h at 650 °C under a constant current density of 250 mW/cm2 is carried out, and the cell with the optimized APS LDC interlayer is stable. Therefore, APS is a viable alternative approach for fabricating an LDC interlayer for SOFCs using the LSGM electrolyte.

Statistical Optimization for the Development of a Culture Medium Based on the Juice of Waste-Dates for Growth of Lactococcus lactis LCL Strain by Using the Plackett–Burman and Response Surface Methodology

The processing industries of dates in Algeria, as elsewhere in other countries, generate very considerable quantities of waste. The purpose of our approach to valorize this dates-waste by using them to achieve at the formulation of a new culture medium for Lactococcus lactis, which is the most widely, used bacteria in the world for most mesophilic dairy starters. A statistical approach was used to investigate the formulation and optimization of a culture medium containing dates juice for the growth of Lactococcus lactis LCL strain. The application of Plackett–Burman screening design was enabled reveals the most significant variables for the biomass production (dates juice, buffer and yeast extract), which were retained for second optimization using a central composite design methodology, leading to determine the optimal concentrations for a maximum growth of Lactococcus lactis LCL strain. The investigated optimal concentrations for a maximum growth were 7% dates juice, 2.6 g/l yeast extract and 250 mM phosphate buffer K2HPO4/NaH2PO4. For these parameters, the maximum cell density of the Lactococcus lactis LCL strain was obtained with a coefficient of determination (R2) of 0.924 for the proposed model, thus demonstrating its robustness. The obtained culture medium in this study containing dates juice represents a favorable medium for the growth of Lactococcus lactis LCL strain improving significantly its growth approximately 30% compared with the M17 medium and that showed a cost about 5 to 6 times lower, than that which is commonly used in the world.

Mild hypothermia upregulates myc and xbp1s expression and improves anti-TNFα production in CHO cells.

Chinese hamster ovary (CHO) cells are the most frequently used host for commercial production of therapeutic proteins. However, their low protein productivity in culture is the main hurdle to overcome. Mild hypothermia has been established as an effective strategy to enhance protein specific productivity, although the causes of such improvement still remain unclear. The self-regulation of global transcriptional regulatory factors, such as Myc and XBP1s, seems to be involved in increased the recombinant protein production at low temperature. This study evaluated the impact of low temperature in CHO cell cultures on myc and xbp1s expression and their effects on culture performance and cell metabolism. Two anti-TNFα producing CHO cell lines were selected considering two distinct phenotypes: i.e. maximum cell growth, (CN1) and maximum specific anti-TNFα production (CN2), and cultured at 37, 33 and 31°C in a batch system. Low temperature led to an increase in the cell viability, the expression of the recombinant anti-TNFα and the production of anti-TNFα both in CN1 and CN2. The higher production of anti-TNFα in CN2 was mainly associated with the large expression of anti-TNFα. Under mild hypothermia myc and xbp1s expression levels were directly correlated to the maximal viable cell density and the specific anti-TNFα productivity, respectively. Moreover, cells showed a simultaneous metabolic shift from production to consumption of lactate and from consumption to production of glutamine, which were exacerbated by reducing culture temperature and coincided with the increased anti-TNFα production. Our current results provide new insights of the regulation of myc and xbp1s in CHO cells at low temperature, and suggest that the presence and magnitude of the metabolic shift might be a relevant metabolic marker of productive cell line.

Butyrated ManNAc analog improves protein expression in Chinese hamster ovary cells.

The chemical additive sodium butyrate (NaBu) has been applied in cell culture media as a direct and convenient method to increase the protein expression in Chinese hamster ovary (CHO) and other mammalian cells. In this study, we examined an alternative chemical additive, 1,3,4-O-Bu3 ManNAc, for its effect on recombinant protein production in CHO. Supplementation with 1,3,4-O-Bu3 ManNAc for two stable CHO cell lines, expressing human erythropoietin or IgG, enhanced protein expression for both products with negligible impact on cell growth, viability, glucose utilization, and lactate accumulation. In contrast, sodium butyrate treatment resulted in a ∼20% decrease in maximal viable cell density and ∼30% decrease in cell viability at the end of cell cultures compared to untreated or 1,3,4-O-Bu3 ManNAc treated CHO cell lines for both products. While NaBu treatment enhanced product yields more than the 1,3,4-O-Bu3 ManNAc treatment, the NaBu treated cells also exhibited higher levels of caspase 3 positive cells using microscopy analysis. Furthermore, the mRNA levels of four cell apoptosis genes (Cul2, BAK, BAX, and BCL2L11) were up-regulated more in sodium butyrate treated wild-type, erythropoietin, or IgG expressing CHO-K1 cell lines while most of the mRNA levels of apoptosis genes in 1,3,4-O-Bu3 ManNAc treated cell lines remained equal or increased only slightly compared to the levels in untreated CHO cell lines. Finally, lectin blot analysis revealed that the 1,3,4-O-Bu3 ManNAc-treated cells displayed higher relative sialylation levels on recombinant EPO, consistent with the effect of the ManNAc component of this additive, compared to control while NaBu treatment led to lower sialylation levels than control, or 1,3,4-O-Bu3 ManNAc-treatment. These findings demonstrate that 1,3,4-O-Bu3 ManNAc has fewer negative effects on cell cytotoxicity and apoptosis, perhaps as a result of a more deliberate uptake and release of the butyrate compounds, while simultaneously increasing the expression of multiple recombinant proteins, and improving the glycosylation characteristics when applied at comparable molarity levels to NaBu. Thus, 1,3,4-O-Bu3 ManNAc represents a highly promising media additive alternative in cell culture for improving protein yields without sacrificing cell mass and product quality in future bioproduction processes.

Quercetin reduces adhesion and inhibits biofilm development by Listeria monocytogenes by reducing the amount of extracellular proteins

The aim of the present study was to investigate the inhibitory mechanisms of quercetin against L. monocytogenes biofilm formation, by affecting secretion of polymeric substances (EPS) and spatio-temporal organization. The minimum concentration of quercetin inhibiting the establishment of bacterial biofilm was determined to be 0.8 mM; therefore, a lower concentration of the flavonoid (0.2 mM) was used in order to investigate its effect on cell attachment and EPS production on stainless steel and the spatio-temporal organization on glass, while avoiding bias of loss of cellular viability. In applying this concentration, reductions of 1.32 and 1.94 Log10 CFU/cm2 of initial adhesion and maximum cell density on stainless steel were observed on the test surfaces at 2 h (initial adhesion) and 24 h (maximum cell density), respectively. EPS on mature biofilms (24 h) was mainly composed by proteins (97.83%), followed by polysaccharides (2.03%) and DNA (0.14%). In addition, quercetin reduced total extracellular protein content by 41% compared with control; while, DNA and polysaccharide contents in biofilms were not affected. Also, relative to control, quercetin impaired surface colonization, early stages of biofilm formation and the development of a more complex architecture during its formation. This proved that quercetin inhibited the formation of L. monocytogenes biofilms by reducing initial attachment, the amount of extracellular proteins found in the EPS matrix and the overall spatio-temporal organization of L. monocytogenes.