Initial Cell Density Research Articles

Escherichia coli survival in plasma‐treated water and in a gas–liquid plasma reactor

AbstractThe effects of post‐plasma exposure to deionized water treated by argon–water plasma and to direct plasma treatment of bacteria in a liquid solution of a strain of Escherichia coli, which was independently evolved to resist the bactericidal effects of hydrogen peroxide, were determined. The hydrogen peroxide resistant phenotype of the evolved strain was proportional to the initial cell density. The catalase activity was significantly elevated in the evolved strain as compared with the wild‐type strain, and the evolved strain resisted the bactericidal effects of plasma‐treated water (post‐plasma exposure) as compared with the wild‐type strain. The evolved strain remained viable after flowing through the plasma reactor and lysed less when exposed to (nonplasma control) hydrogen peroxide solutions as compared with the wild‐type cells.

Improving Fucoxanthin Production in Mixotrophic Culture of Marine Diatom Phaeodactylum tricornutum by LED Light Shift and Nitrogen Supplementation.

Fucoxanthin (Fx), a kind of primary carotenoids in brown seaweeds and diatoms, has attractive efficacy in human’s healthcare including loss weight, the prevention of diabetes and Alzheimer’s disease. Marine diatom Phaeodactylum tricornutum is now realized as a promising producer for commercial Fx production due to its higher content of Fx than brown seaweeds with easily artificial cultivation and Fx extraction. In the present study, to improve Fx production in P. tricornutum, the mixotrophic cultures were applied to optimize initial cell density, light intensity, light regime and nitrogen supplementation. The results showed that the higher initial cell density (1 × 107 cells mL–1) and lower light intensity (20 μmol m–2 s–1) were favorable for biomass production and Fx accumulation. The maximal Fx content [16.28 mg g–1 dry weight (DW)] could be achieved under blue light (BL), but the highest biomass concentration (5.53 g L–1) could be attained under red: blue light (R: B, 6:1) in the batch culture. A novel two-phase culture approach was developed to increase the biomass concentration to the highest value (6.52 g L–1) with the maximal productivity of Fx (8.22 mg L–1 d–1) through light shift from R:B ratio (6:1) in phase 1 to R:B ratio (5:1) by enhancing BL and tryptone addition in phase 2. The content and intracellular amount of Fx were also increased 8% and 12% in phase 2 compared to phase 1. The expression levels analysis revealed that genes encoding phytoene synthase (PSY), zeaxanthin epoxidase (ZEP), and fucoxanthin-chlorophyll-protein b (FCPb) were upregulated significantly, with downregulation of the gene encoding violaxanthin de-epoxidase (VDE), leading to the improvement of Fx in phase 2. The present study demonstrated the two-phase culture strategy could promote Fx productivity through enhancing biomass production and increasing Fx content, indicating that strengthening BL coupled with adding tryptone were effective to facilitate Fx production by mixotrophic cultivation of marine diatom P. tricornutum.

Homogenization of initial cell distribution by secondary flow of medium improves cell culture efficiency

Homogenization of the initial cell distribution is essential for effective cell development. However, there are few previous reports on efficient cell seeding methods, even though the initial cell distribution has a large effect on cell proliferation. Dense cell regions have an inverse impact on cell development, known as contact inhibition. In this study, we developed a method to homogenize the cell seeding density using secondary flow, or Ekman transportation, induced by orbital movement of the culture dish. We developed an orbital shaker device that can stir the medium in a 35-mm culture dish by shaking the dish along a circular orbit with 2 mm of eccentricity. The distribution of cells in the culture dish can be controlled by the rotational speed of the orbital shaker, enabling dispersion of the initial cell distribution. The experimental results indicated that the cell density became most homogeneous at 61 rpm. We further evaluated the cell proliferation after homogenization of the initial cell density at 61 rpm. The results revealed 36% higher proliferation for the stirred samples compared with the non-stirred control samples. The present findings indicate that homogenization of the initial cell density by Ekman transportation contributes to the achievement of higher cell proliferation.

Xylose 기질을 소모하는 재조합 Saccharomyces cerevisiae의 lactic acid 생산수율 향상을 위한 조건 탐색

Lactic acid is widely used in the food, pharmaceutical, textile and chemical industries. Also, poly-lactic acid (PLA) is in increasing demand as an environmentally friendly and biodegradable plastic. In our prior study, a lactic acid-producing, xylose-fermenting Saccharomyces cerevisiae strain (SR8 LDH) has been developed. In the present study, we tested the SR8 LDH strain under various fermentation conditions to discover the most important condition determining lactic acid and ethanol production profiles. Using xylose as a substrate significantly improved lactic acid yields compared to using glucose under microaerobic conditions. High initial cell density improved lactic acid productivity, but it did not affect ethanol production. Lastly, when fermenting xylose, the PDC1 gene encoding pyruvate decarboxylase was transcriptionally repressed, which might be associated with the production of low ethanol and high lactic acid. These results indicate that the type of a carbon source, i.e. using xylose instead of glucose could be a promising solution for lactic acid production by engineered S. cerevisiae.

Functional Expression of Adenosine A3 Receptor in Yeast Utilizing a Chimera with the A2AR C-Terminus.

The adenosine A3 receptor (A3R) is the only adenosine receptor subtype to be overexpressed in inflammatory and cancer cells and therefore is considered a novel and promising therapeutic target for inflammatory diseases and cancer. Heterologous expression of A3R at levels to allow biophysical characterization is a major bottleneck in structure-guided drug discovery efforts. Here, we apply protein engineering using chimeric receptors to improve expression and activity in yeast. Previously we had reported improved expression and trafficking of the chimeric A1R variant using a similar approach. In this report, we constructed chimeric A3/A2AR comprising the N-terminus and transmembrane domains from A3R (residues 1–284) and the cytoplasmic C-terminus of the A2AR (residues 291–412). The chimeric receptor showed approximately 2-fold improved expression with a 2-fold decreased unfolded protein response when compared to wild type A3R. Moreover, by varying culture conditions such as initial cell density and induction temperature a further 1.7-fold increase in total receptor yields was obtained. We observed native-like coupling of the chimeric receptor to Gai-Gpa1 in engineered yeast strains, activating the downstream, modified MAPK pathway. This strategy of utilizing chimeric receptor variants in yeast thus provides an exciting opportunity to improve expression and activity of “difficult-to-express” receptors, expanding the opportunity for utilizing yeast in drug discovery.

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.

Tissue-in-a-Tube: three-dimensional in vitro tissue constructs with integrated multimodal environmental stimulation.

Three-dimensional (3D) in vitro tissue models are superior to two-dimensional (2D) cell cultures in replicating natural physiological/pathological conditions by recreating the cellular and cell-matrix interactions more faithfully. Nevertheless, current 3D models lack either the rich multicellular environment or fail to provide appropriate biophysical stimuli both of which are required to properly recapitulate the dynamic in vivo microenvironment of tissues and organs. Here, we describe the rapid construction of multicellular, tubular tissue constructs termed Tissue-in-a-Tube using self-assembly process in tubular molds with the ability to incorporate a variety of biophysical stimuli such as electrical field, mechanical deformation, and shear force of the fluid flow. Unlike other approaches, this method is simple, requires only oxygen permeable silicone tubing that molds the tissue construct and thin stainless-steel pins inserted in it to anchor the construct and could be used to provide electrical and mechanical stimuli, simultaneously. The annular region between the tissue construct and the tubing is used for perfusion. Highly stable, macroscale, and robust constructs anchored to the pins form as a result of self-assembly of the extracellular matrix (ECM) and cells in the bioink that is filled into the tubing. We demonstrate patterning of grafts containing cell types in the constructs in axial and radial modes with clear interface and continuity between the layers. Different environmental factors affecting cell behavior such as compactness of the structure and size of the constructs can be controlled through parameters such as initial cell density, ECM content, tubing size, as well as the distance between anchor pins. Using connectors, network of tubing can be assembled to create complex macrostructured tissues (centimeters length) such as fibers that are bifurcated or columns with different axial thicknesses which can then be used as building blocks for biomimetic constructs or tissue regeneration. The method is versatile and compatible with various cell types including endothelial, epithelial, skeletal muscle cells, osteoblast cells, and neuronal cells. As an example, long mature skeletal muscle and neuronal fibers as well as bone constructs were fabricated with cellular alignment dictated by the applied electrical field. The versatility, speed, and low cost of this method is suited for widespread application in tissue engineering and regenerative medicine.

Electron transfer via the non-Mtr respiratory pathway from Shewanella putrefaciens CN-32 for methyl orange bioreduction

Exoelectrogens play the core roles in bioelectrochemical systems (BESs) because of their unique extracellular electron transfer capacity to different electron acceptors. Microbial reduction of azo dyes by exoelectrogens under anaerobic conditions has received great attention because of its eco-friendliness, low cost, and unique extracellular reduction ability. In this work, we unexpectedly found that Shewanella putrefaciens CN-32 adopted a distinctive electron transfer mechanism for bioreduction of MO (methyl orange) compared to the other exoelectrogens. MO reduction by S. putrefaciens CN-32 occurred through mechanisms that were not dependent on the known azoreductase and the Mtr (metal-reducing) respiratory pathway. Some anaerobic regulators (e.g., Fur and EtrA) and periplasmic c-type cytochromes (Sputcn32_2333) might involve in MO reduction by S. putrefaciens CN-32. The major reduction products were 4-aminobenzenesulfonic acid (4-ABA) and N, N-dimethyl-p-phenylenediamine (DPD) and the initial cell density in the reduction system affected MO reduction kinetics by S. putrefaciens CN-32. Moreover, S. putrefaciens CN-32 could utilize multiple mediators such as flavins or anthraquinone-2,6-sodium disulfonate (AQDS) to accelerate MO reduction. Our findings provide a new perspective on the reduction mechanisms of azo dyes by exoelectrogens and might facilitate more efficient utilization of them in BESs for treatments of azo dyes-polluted industrial effluents.

High-yield production of biomass, protein and pigments by mixotrophic Chlorella pyrenoidosa through the bioconversion of high ammonium in wastewater

To achieve a high consumption rate of ammonium with biomass coproduction, the mixotroph Chlorella pyrenoidosa was cultivated in high ammonium-high salinity wastewater medium in this study. The initial cell density, glucose and ammonium concentrations, and light intensity were optimized in shake flasks. A 5-L fermenter with surrounding LED (Light Emitting Diode) and a 50-L fermenter with inlet LED were employed for batch and semicontinuous cultivation. The results demonstrated that the highest contents of protein (56.7% DW) and total pigments (4.48% DW) with productivities of 5.62 g L-1 d-1 and 0.55 mg L-1 d-1, respectively, were obtained in 5-L photofermenter, while the maximum NH4+ consumption rate (1,800 mg L-1 d-1) and biomass yield (23.6 g L-1) were achieved in 50-L photofermenter. This study developed a novel strategy to convert high ammonium in wastewater to high-protein algal biomass, facilitating wastewater bioremediation and nitrogen recycling utilization by the mixotroph C. pyrenoidosa in photofermentation.

Effect of different irradiation doses on human and murine fibroblasts used as feeder layer for keratinocytes’ culture

Background & Aim The establishment of human keratinocytes’ culture (KTs) to obtain human Tissue Engineered Skin Substitutes is under research nowadays. Best results have been achieved culturing KTs on a feeder layer of irradiated 3T3 murine fibroblasts (i3T3s). Nevertheless, molecular effects of murine cells on KTs is unknown and for this reason, irradiated human fibroblasts (iHFs) have been studied. The objective of this study is to evaluate the effect of different irradiation doses on cell reducing power and cell proliferation of both fibroblasts. Methods, Results & Conclusion Methods 3T3s were obtained from a commercial vial of 3T3-Swiss Albino. HFs were isolated from human skin biopsies. After culturing and final recovery, both cell types were irradiated to doses of 50 and 100 Gy to constitute the feeder. Non-irradiated cells were used as control. Each condition was cultured 15 days in two 24-well plates (2 cm2/well). In each plate, cell densities cultured in triplicate were: 7,500 cells/cm2 and 10,000 cells/cm2. One plate was used for reducing power analysis; using a dilution of PrestoBlue™ (10%). The other plate was used for cell proliferation analysis; using Trypan Blue solution. N for each plate was 3 and a value of p Results Analysis of each type of feeder (Figs. 1-2) revealed significant differences at Days 10 and 15, between both irradiation doses and non-irradiated cells. In the case of iHFs, irradiation dose had a significant effect on cell reducing power, mainly at Day 15. Analysis of i3T3s revealed that the effect of irradiation on reducing power was extremely significant from Day 3 to 15, but no dose-dependent. A comparative analysis (Fig. 3) indicated that the number of non-irradiated cells after 15 days was higher in the case of 3T3s than HFs (644,620 cells vs 143,740 cells), but cell reducing power was lower in the case of murine fibroblasts (72.33% vs 80.67%). In the case of irradiated cells, iHFs were more resistant to irradiation in both, cell reducing power (27.5% vs 4.92%) and cell proliferation (5,962 cells vs 1,351 cells) at Day 15. Conclusion No differences exist considering initial cell density or irradiation dose in both feeders. Comparative analysis revealed that irradiation process is more effective in 3T3s than in HFs. This study needs to be completed with the incorporation of KTs on the different feeder conditions analyzed. Supported by PI17/02083, SAS PI-0458-2016 and PhD fellowship FI18/00269–Alvaro Sierra-Sanchez).

A correlation study on optimum conditions of microbial precipitation and prerequisites for self-healing concrete

Microbial induced CaCO3 precipitation (MICP) based upon enzymatic urea hydrolysis has been verified as an effective way for crack treatment, especially for self-healing of concrete cracks. This paper aimed at correlating optimum conditions of MICP with prerequisites for self-healing concrete. Orthogonal experiments on a combination of factors contributing to the MICP process were firstly performed. Initial cell density and Ca2+ concentration were highly significant factor and significant factor respectively. High initial cell density (1×108 cells·mL-1) together with relatively low Ca2+ concentration (50 mM) favored microbial precipitation. The second part of this study was associated with dissolution tests to simulate the dissolving behavior of urea and calcium, since the dissolving of healing agents in cracks is a prerequisite of self-healing. By an addition of urea and Ca(NO3)2 with constant mass ratio of 2:3 in concrete, the highest values of the estimated urea concentration (345 mM) and Ca2+ concentration (44 mM) dissolved in cracks were close to the optimal values found by orthogonal studies. Although the addition of urea and Ca(NO3)2 would not have a negative impact on the mechanical properties of concrete, direct mixing is not recommended due to the low utilization efficiency of incorporating healing agents for self-healing.

Investigation of fermentation conditions of biodiesel by-products for high production of β-farnesene by an engineered Escherichia coli.

Recently, the research on conversion of biodiesel by-products to high value-added products has received much attention, due to the adverse effects of large accumulations of biodiesel by-products caused by the rapid increase in biodiesel production. Herein, this study investigated the utilization of by-products crude glycerol (CG-1 and CG-2) from two different industrial methods of biodiesel production and the favorable fermentation conditions for the high yield of β-farnesene by an engineered Escherichia coli F4, which harbored an optimized mevalonate pathway. Through analyzing by-products' components and fermentation performance, we found that CG-2 did not contain harmful impurities such as methanol and black solid impurities, and the β-farnesene production was up to 2.7g/L from CG-2, which was similar to that from pure glycerol (2.5g/L) and higher than that (2.21g/L) from CG-1. Therefore, CG-2 was more suitable for β-farnesene production than CG-1, which might provide a reference for choosing a more suitable method on practical biodiesel production. Afterward, a variety of important fermentation conditions were explored using CG-2 as a substrate in shaken flasks. Under the optimal conditions (including induced cell density 1.0, initial cell density 0.25, temperature after induction 33°C, initial medium pH6.5), the yield of β-farnesene from CG-2 reached 10.31g/L in a 5-L bioreactor, which was 2.8-fold higher than initial conditions in shake flasks and was the highest yield of β-farnesene produced from biodiesel by-products by fermentation as well. The recommended fermentation conditions in this work will provide a valuable reference for the industrial production of β-farnesene utilizing biodiesel by-products.

Interspecific competition between Cylindrospermopsis raciborskii and Microcystis aeruginosa on different phosphorus substrates.

Phosphorus (P) is responsible for algal growth and the structural changes in algal communities. Therefore, it is essential to know whether the different phosphorus availability to different algae can change the community structure. In this study, the interspecific competition was investigated at two bloom-forming cyanobacterium, Cylindrospermopsis raciborskii and Microcystis aeruginosa, when both were treated with five different phosphate compounds, including K2HPO4, β-glycerol phosphate, (2-aminoethyl)-phosphinic acid, glyphosate, and P-free. The results of mono-culture experiments showed that the two species could utilize the dissolved organic phosphorus (DOP) and K2HPO4 (DIP) as the sole P resource. Moreover, the specific growth rates and the endogenous alkaline phosphatase activity in M. aeruginosa cells were much lower than those in C. raciborskii under DOP and DIP treatments. In the co-cultured experiments, however, a significant biomass increase in C. raciborskii was observed in all experimental P treatments, except for glyphosate, regardless of its initial cell density proportion. A 31.8-63.4% increase in cell number of C. raciborskii was found after incubated into K2HPO4, while the highest biomass of mixed samples, 17.72 × 106 cell mL-1, was observed in the (2-aminoethyl)-phosphinic acid treatment (50C50M). Additionally, higher specific growth rate was also found in C. raciborskii when compared with M. aeruginosa under P-free; the increasing proportion of C. raciborskii were 29.1% (50C50M), 16.4% (75C25M), and 36.7% (25C75M), respectively. When the mixed samples were co-cultivated under glyphosate, C. raciborskii cells appeared to be depressed, whereas the cell density of M. aeruginosa increased rapidly. The findings indicated that an excellent P competition might give some advantages for C. raciborskii dominance in natural waters with DIP limitation or DOP abundance.

Identifying density-dependent interactions in collective cell behaviour.

Scratch assays are routinely used to study collective cell behaviour in vitro. Typical experimental protocols do not vary the initial density of cells, and typical mathematical modelling approaches describe cell motility and proliferation based on assumptions of linear diffusion and logistic growth. Jin et al. (Jin et al. 2016 J. Theor. Biol.390, 136-145 (doi:10.1016/j.jtbi.2015.10.040)) find that the behaviour of cells in scratch assays is density-dependent, and show that standard modelling approaches cannot simultaneously describe data initiated across a range of initial densities. To address this limitation, we calibrate an individual-based model to scratch assay data across a large range of initial densities. Our model allows proliferation, motility, and a direction bias to depend on interactions between neighbouring cells. By considering a hierarchy of models where we systematically and sequentially remove interactions, we perform model selection analysis to identify the minimum interactions required for the model to simultaneously describe data across all initial densities. The calibrated model is able to match the experimental data across all densities using a single parameter distribution, and captures details about the spatial structure of cells. Our results provide strong evidence to suggest that motility is density-dependent in these experiments. On the other hand, we do not see the effect of crowding on proliferation in these experiments. These results are significant as they are precisely the opposite of the assumptions in standard continuum models, such as the Fisher-Kolmogorov equation and its generalizations.

Optimization of production conditions of rice α-galactosidase II displayed on yeast cell surface

The yeast surface displayed rice α-galactosidase II (YSD rice α-Gal II) was generated with the pYD1 vector. The expression and cultural conditions for the improvement of production of YSD rice α-Gal II were optimized. The results showed that several induction factors, which were the initial cell density, inoculation ratio, galactose (inducer) concentration, induction time and temperature, determined the activity and expression efficiency of YSD rice α-Gal II. Meanwhile, the medium composition also affected its activity and production. Moreover, the production of YSD rice α-Gal II was further improved by continuous feeding of galactose in the fermenter level. The highest production was obtained at an initial cell density of OD600 = 2.9, 2% inoculation ratio, and 2% galactose, with 0.6 g/L compound nitrogen source ((NH4)2SO4/urea = 2/1, w/w) and 5 g/L sucrose, followed by continuous feeding of galactose (20 g/L with flow rate of 1.5 mL/h). At such conditions, the enzyme activity and productivity reached to 676.2 U/g (DCW) and 1548.5 U/L, respectively, 26.4- and 63.7-fold to that before optimization. The results provided a basic and effective strategy for the industrial production of YSD rice α-Gal II.

Rapid process development of serum-free pseudorabies virus production with the Quality by Design approach.

This study described a successful application of the Quality by Design (QbD) approach to pseudorabies virus (PRV) production process development in a fixed-bed bioreactor using the serum-free medium (SFM). The innovated tube-fixed-bed bioreactor was used as a scale-down model of the fixed-bed bioreactor for process development. Risk analysis was performed using Ishikawa diagram combined with failure mode effects analysis (FMEA). The comparative experiment was performed to screen proper medium for adherent African green monkey kidney (Vero) cells from three commercially available SFMs (VP-SFM, ProVERO-1 and Vero-A). The Vero-A medium showed as an outstanding one for further study. The PRV titer in harvest medium was consider as Critical Quality Attribute (CQA) and the Critical Process Parameters (CPPs) [time of infection (TOI), multiplicity of infection (MOI) and initial inoculation cell density] ranked high with risk priority number (RPN) were taken into design of experiment (DoE) methodology. Then prediction model of PRV production process was established and a robust PRV production process was explored. Under the robust setpoint conditions, the Xcell 1L laboratory-scale fixed-bed bioreactor yielded PRV titer up to 7.87 log10 TCID50/mL at 3dpi, which was comparable with that in the tube-fixed-bed bioreactor. Combination of the tube-fixed-bed bioreactor and QbD approach could further accelerate the development of a robust virus production process.

Hydrogen Production from Methane by Methylomonas sp. DH-1 under Micro-aerobic Conditions

Fueled by the recognition of hydrogen as a promising renewable energy source for the future, there have been many attempts to find greener and more economical ways for its production from various sources. In this study, Methylomonas sp. DH-1, a type I methanotroph, was found to produce hydrogen using methane as a sole carbon source, under micro-aerobic conditions; this is analogous to the partial oxidation of methane in a thermochemical process based on metal catalysts. Flask cultures of Methylomonas sp. DH-1 were used to investigate the effects of different culture conditions on hydrogen production, including oxygen levels, methane/oxygen ratios, and initial cell densities. Methylomonas sp. DH-1 could produce hydrogen at an oxygen level below 4%, regardless of the methane content in the flask, implying that the critical factor for hydrogen production is the oxygen level, rather than the methane/oxygen ratio. Moreover, Methylomonas sp. DH-1 shows reversibility in hydrogen production and uptake, because the strain produces hydrogen under micro-aerobic conditions, uptakes it when the oxygen levels increase, and restores the hydrogen production capability when conditions become microaerobic again. Under initial conditions of 30% methane, 70% air, and an OD600nm of 6, hydrogen production was 26.87 μmol and its yields per methane and dry cell weight were 14.98 mmol-H2/mol-CH4 and 101.53 μmol-H2/g DCW, respectively, after 24 h of cultivation.

Specific oceanographic characteristics and phytoplankton responses influencing the primary production around the Ulleung Basin area in spring

The East Sea (Sea of Japan) is a marginal, semi-closed sea in the northwestern Pacific. The Ulleung Basin area, which is located near the subpolar front of the East Sea, is known to have high primary production and good fisheries in spring season. After episodic wind-driven events during the spring of 2017, horizontal and vertical profiles of physical chemical biological factors were investigated at 29 stations located in the Ulleung Basin area. In addition, growth responses of phytoplankton communities to nutrient additions were evaluated by bioassay experiments to understand the fluctuation of phytoplankton biomass. Because of strong northwestern wind, phytoplankton biomass was scattered and upwelling phenomenon might be suppressed in this season. The phytoplankton abundances in the coastal stations were significantly higher than offshore and island stations. In contrast, the nutrient and chlorophyll a (Chl a) concentrations and the phytoplankton biomass were quite low in all locations. Bacillariophyceae was dominated group (75.1% for coastal, 40.0% for offshore and 43.6% for island stations). In the algal bioassays, the phytoplankton production was stimulated by N availability. The in vivo Chl a values in the +N and +NP treatments were significantly higher than the values in the control and the +P treatments. Based on the field survey, the higher nutrients in coastal waters affected the growth of diatom assemblages, however, little prosperity of phytoplankton was observed in the offshore waters despite the injection of sufficient nutrients in bioassay experiments. The growth of phytoplankton depended on the initial cell density. All of results indicated that a dominant northwestern wind led to a limited nutrients condition at euphotic layers, and the low level of biomass supply from the coasts resulted in low primary production. Both supplying nutrients and introducing phytoplankton through the currents are critical to maintain the high productivity in the Ulleung Basin area of the East Sea.

Competitive interactions between two allelopathic algal species: Heterosigma akashiwo and Phaeodactylum tricornutum

ABSTRACTBoth Heterosigma akashiwo and Phaeodactylum tricornutum have been reported to produce allelochemicals capable of inhibiting the growth of co-occurring microalgae. Here, potential allelopathy between H. akashiwo and P. tricornutum was evaluated using bi-algal culture, cell-free culture filtrate and a no-contact co-culturing system in nutrient-replete media. Experiments were also conducted in the no-contact co-culturing system under nutrient-limited conditions. In nutrient-replete bi-algal culture, the growth of P. tricornutum and H. akashiwo each tended to be strongly suppressed when the other species was inoculated at high cell densities. A mathematical model was used to simulate the growth interactions of the two species in bi-algal culture and showed that P. tricornutum outcompeted H. akashiwo over time with different initial cell densities under nutrient-sufficient conditions, indicating that P. tricornutum was more potent in allelopathy. Heterosigma akashiwo growth was inhibited both in the P. tricornutum culture filtrate and no-contact co-culturing system. This confirmed that the extracellular allelopathic compounds released by P. tricornutum were one of the sources of the H. akashiwo growth inhibition. Nutrient deficiency did not increase the extent of allelopathic activity of allelochemicals.

In-Situ Stable Injectable Collagen-Based Hydrogels for Cell and Growth Factor Delivery

Here we report development of in-situ stable injectable hydrogels for delivery of cells and growth factors based on two precursors, alginate, and collagen/calcium sulfate (CaSO4). The alg/col hydrogels were shear-thinning, injectable through commercially available needles and stable right after injection. Rheological measurements revealed that pre-crosslinked alg/col hydrogels fully crosslinked at 37°C and that the storage modulus of alg/col hydrogels increased with increasing the collagen content or the concentration of CaSO4. The viscoelastic characteristics and injectability of the alg/col hydrogels were not significantly impacted by the storage of precursor solutions for 28 days. An osteo-inductive bone morphogenic protein-2 (BMP-2) loaded into alg/col hydrogels was released in 14 days. Human mesenchymal stem cells (hMSCs) encapsulated in alg/col hydrogels had over 90% viability over 7 days after injection. The DNA content of hMSC-laden alg/col hydrogels increased by 6-37 folds for 28 days, depending on the initial cell density. In addition, hMSCs encapsulated in alg/col hydrogels and incubated in osteogenic medium were osteogenically differentiated and formed a mineralized matrix. Finally, a BMP-2 loaded alg/col hydrogel was used to heal a critical size calvarial bone defect in rats after 8 weeks of injection. The alg/col hydrogel holds great promise in tissue engineering and bioprinting applications.