Maximum Cell Density Research Articles

Design, experimental optimization, and flow analysis of a novel bioreactor for dynamic mammalian cell culture at laboratory scale using Box-Behnken design

Improving the quality of dynamic cell culture in laboratories is an important field in bioengineering. In this study, a novel lab-scale bioreactor using a vibrating agitator and a modified flask has been introduced to create a strong mixing at low shear stress. This bioreactor has been optimized using Box-Behnken design based on three dimensionless important structural factors including disc diameter, vibration amplitude, and the height of the disc placement. Three growth indicators including the specific growth rate, the natural logarithm of the maximum cell density, and productivity have been considered as biological responses. The results show that the disc diameter has the most important role in these indicators. If the disc diameter, vibration amplitude, and the height of disc placement are set to 0.24, 0.02, and 0.4 of the flask diameter, respectively, the values of the specific growth rate, the maximum cell density, and productivity at this optimum settings are 0.033 (h−1), 13.11, and 5133 (cells/(mL.h)), respectively. These values of the indicators are high and indicate the better performance of this bioreactor than other lab-scale bioreactors. In addition, investigating Reynolds number in the fluid flow indicates that in the range of 780 up to 1150, growth indices are high.

Escherichia coli Survival on Dry Bulb Onions Treated with Crop Protection Sprays Prepared using Contaminated Water in the Treasure Valley Growing Region

Contaminated agricultural water has been implicated in produce-associated outbreaks, including dry bulb onions (Allium cepa). This study was designed to quantify risks associated with t contaminated water used to prepare crop protection sprays applied immediately before the onset of field curing of dry bulb onions. Laboratory experiments determining the behavior of Salmonella and Escherichia coli in crop protection chemical solutions were performed to guide selection for field use. Field trials were conducted (2022, 2023) in eastern Oregon (Treasure Valley) using two onion cultivars (‘Red Wing’ and ‘Cometa’) inoculated with a rifampicin-resistant E. coli cocktail (3–4 log CFU/100 mL) suspended in fungicide solution or clay suspension, and applied with a backpack sprayer at the end of the growing season. Onions were sampled through the next 4 weeks of field curing and after 1 and 4–5 mos of postharvest storage. In 2022, onions were initially contaminated at a maximum cell density of 48 MPN/onion (Geometric mean (GM): 3.7 MPN/onion). At the end of curing, a single onion (out of 320) tested positive at 2 MPN/onion. In 2022, E. coli was not detected during postharvest storage (n = 160). In 2023, the application of contaminated sprays resulted in a maximum contamination of 275 MPN/onion (GM: 8.6 MPN/onion). At the end of the 2023 curing period, three out of 320 onions (0.9%) had detectable levels of E. coli (1–2 MPN/onion). Three ‘Cometa’ onions from the same plot that were treated with fungicide were positive for E. coli after 5 months of postharvest storage (2, 11, and 83 MPN/onion). These field trials indicate field curing conditions in the Treasure Valley help mitigate risks associated with contaminated water used for applying crop protection sprays. E. coli was detected on a small percentage of onions at low cell density after curing. The single onion with elevated E. coli populations after postharvest storage had internal damage characteristic of bacterial rot.

Effect of Ca doping at A or B sites on electrochemical performance of La1.9In0.1Ce2O7 proton conductors

Fluorite proton conductors La1.8In0.1Ca0.1Ce2O7-δ and La1.9In0.1Ce1.9Ca0.1O7-δ (LICaC and LICCa) were synthesized by solid state reaction. The effect of Ca doping at A site or B site on La1.9In0.1Ce2O7 was studied by X-ray diffraction (XRD), Inductively Coupled Plasma Mass Spectrometry (ICP), scanning electron microscopy (SEM) and electrical conductivity measurement. XRD shows that the LICaC and LICCa were successfully synthesized, and Ca was doped to the expected sites. The measured mass ratios of each element by ICP were in good agreement with their theoretical values. In addition, SEM also found that the surface of the sample was dense without holes. The conductivities of LICaC and LICCa were studied at 400–800 ℃, and their highest conductivities were 7.28×10−4 and 5.56×10−3 S·cm−1, respectively. The conductivity of LICCa doped at the B site is better than that of LICaC doped at the A site. In the wet air at 800 ℃, the proton transference numbers of LICaC and LICCa are 0.5 and 0.52, respectively. LICaC and LICCa were used as electrolytes to prepare fuel cells and their performance was tested. The electrolyte thickness of LICAC and LICCA was about 0.9 mm. At 700 ℃, the maximum power density of LICaC and LICCa electrolyte-based single cells are 124.51 and 162.26 mW cm−2, respectively. The results show that LICCa has good proton conductivity and has potential as an electrolyte for high-performance fuel cells.

Fabrication of stable slippery lubricant-infused porous surface on polymethyl methacrylate/thermoplastic polyurethane by supercritical CO2 foaming

The development of sustainable and efficient methods to prepare slippery lubricant-infused porous surface (SLIPS) is a profound work. In this study, using bilayer polymers restricting foaming mutually, bimodal cells were prepared through bilayer poly(methyl methacrylate) (PMMA). At the same time, uniform cells were prepared by bilayer PMMA /thermoplastic polyurethane (TPU). The prepared porous surfaces exhibited a high porosity (57% or more). TPU as dispersed phase increased the cell density of the PMMA/TPU surface with a maximum cell density of 5.5 × 107 cells/cm2 and an average cell size of 1.0 μm. SLIPS prepared on PMMA/TPU surface with high porosity and uniform microcellular had better stability, and the sliding angle (SA) remained less than 10° after centrifugal rotation at 8000r/min. Therefore, this work provides an approach to improve the surface cell density and produce SLIPS sustainably and efficiently.

Application of a microalga, Tetradesmus obliquus PF3, for NO and CO2 removal from actual flue gas via cultivating in wastewater.

Greenhouse gas (GHG) emissions, particularly anthropogenic emissions, are the primary drivers of climate change. The cultivation of microalgae represents a highly promising strategy for mitigating atmospheric GHG levels. The growth characteristics and GHG mitigation capabilities of Tetradesmus obliquus PF3 were investigated in domestic wastewater at a thermal power plant. The maximum cell density and productivity were 1.52 ± 0.01 g L-1 and 0.33 ± 0.01 g L-1 day-1, respectively. Utilizing a serial configuration of two reactors, the elimination efficiency of NO and CO2 attained values of 78 ± 4% and 14 ± 4%, respectively. NO concentration at the outlet was less than 24.6 ± 2.9 mg m-3, meeting the latest Chinese discharge limits. Besides, the recovery efficiency of NO and CO2 increased to 77 ± 8% and 2.24 ± 0.04%, respectively, compared to that of the single reactor (40 ± 3%, 0.9 ± 0.0%). A removal efficiency of over 90% was achieved for TN and TP in domestic wastewater. The concentrations of COD (76.5 mg L-1), NH4+-N (0.9 mg L-1), TN(6.31 mg L-1), and TP (0.35 mg L-1) in effluent were below the thresholds of 100 mg L-1, 25 mg L-1, none data, and 3 mg L-1, respectively, complying with the Chinese Discharge Standard (Class II criteria set forth) for Municipal Wastewater Treatment Plants Pollutants. The harvested biomass exhibited a high content of carbohydrates and proteins, making it a viable feedstock for biofuels and bio-fertilizers. Our results demonstrate that Tetradesmus obliquus PF3-based flue gas treatment technology can simultaneously realize GHG removal, wastewater bio-remediation, and biomass recovery.

Effect of inorganic nanoparticles on polypropylene in‐mold decoration and microcellular foaming injection molding composites

AbstractIn this paper, the surface morphology of the parts was improved by the in‐mold decoration and microcellular foaming injection molding (MIM‐IMD), and the mechanical properties of the parts were improved by inorganic particles modification. The effects of different inorganic particles (CaCO3, MMT, Talc) on the rheological behavior, crystallization properties, cell structure, surface quality, and mechanical properties of the MIM‐IMD composites were analyzed through experimental research and comprehensive macro and micro characterization. It was found that the foamed composite added with MMT had the highest surface quality, the best cell structure, the minimum average cell diameter of 78.4 μm and the maximum cell density of 3.09 × 106 cells/cm3. Talc improved the crystallinity of polypropylene (PP) matrix, and the resulting mechanical properties were the most significant improvement. Tensile, flexural, and impact strength were increased by 20.3%, 11.6%, and 45.2%, respectively.Highlights The MIM‐IMD process was used to improve the surface quality of the samples. The enhancement of different inorganic particles on the properties were compared. The properties were evaluated by macro‐ and micro‐characterization. Inorganic particles can significantly improve the cell structure. Inorganic particles can greatly improve mechanical properties.

Biological effects of culture medium on Tetraselmis chuii and Dunaliella tertiolecta: Implications for emerging pollutants degradation

In this study, laboratory-scale cultivation of T. chuii and D. tertiolecta was conducted using Conway, F/2, and TMRL media to evaluate their biochemical composition and economic costs. The highest cell density (30.36 × 106 cells/mL) and dry weight (0.65 g/L) for T. chuii were achieved with Conway medium. This medium also produced biomass with maximum lipid content (25.65%), proteins (27.84%), and total carbohydrates (8.45%) compared with F/2 and TMRL media. D. tertiolecta reached a maximum cell density of 17.50 × 106 cells/mL in F/2 medium, which was notably lower than that of T. chuii. Furthermore, the media cost varied from US$0.23 to US$0.74 for each 1 L of media, primarily due to the addition of Na3PO4, KNO3, and cyanocobalamin. Thus, biomass production rates varied between US$38.81 and US$128.80 per kg on a dry weight basis. These findings comprehensively compare laboratory conditions and the costs associated with biomass production in different media. Additionally, this study explored the potential of T. chuii and D. tertiolecta strains, as well as their consortia with bacteria, for the degradation of various emerging pollutants (EPs), including caffeine, salicylic acid, DEET, imidacloprid, MBT, cimetidine, venlafaxine, methylparaben, thiabendazole, and paracetamol. Both microalgal strains demonstrated effective degradation of EPs, with enhanced degradation observed in microalgae-bacterial consortia. These results suggest that the symbiotic relationship between microalgae and bacteria can be harnessed for the bioremediation of EPs, thereby offering valuable insights into the environmental applications of microalgal cultivation.

Assessment of Nutrient Removal Efficiency of Marine Microalgae Dunaliella salina in Saline Food Industry Wastewater

Industrial wastewater release into the environment is a critical global challenge leading to severe water pollution, adversely impacting terrestrial and aquatic ecosystems requiring effective solutions. The treatment, disposal, and recirculation of saline wastewater from food containing nitrate and phosphate poses significant health and environmental risks. This research aims to address the challenge by investigating the potential of using the marine microalgae Dunaliella salina for treating saline food industry wastewater, both in laboratory and pilot-scale settings using raceway tanks. In the laboratory study, a mixture of wastewater and F/2 medium was inoculated with Dunaliella salina to assess its potential for growth in saline wastewater, rich in nitrate and phosphate. The study observed a cell density of 5.5 x 105 cells/ml. Subsequently, a pilot-scale study was conducted, where the maximum cell density of 6.2 x 105 cells/ml was achieved. The efficacy of Dunaliella salina in removing nitrate and phosphorus from the culture was evaluated by measuring the levels of these compounds. A significant reduction in nitrate concentration from 146 to 22.16 mg/l was observed, with a removal efficiency of 84.82%; similarly, the phosphate concentration decreased from 55.3 to 10.79 mg/l, with a removal efficiency of 80.49 %. The reductions in nitrate and phosphate concentrations, along with the demonstrated growth of Dunaliella salina in saline industrial wastewater, confirmed the feasibility of employing these microalgae for bioremediation treatment in a sustainable way.

Adeno-associated virus perfusion enhanced expression: A commercially scalable, high titer, high quality producer cell line process

With safety and efficacy demonstrated over hundreds of clinical trials in the last 30 years, along with at least six recent global marketing authorizations achieved since 2017, recombinant adeno-associated viruses (rAAVs) have been established asthe leading therapeutic gene transfer vector for rare, monogenic diseases. Significant advances in manufacturing technology have been made in the last few decades to address challenges with GMP production of rAAV products, although yield, cost, scalability, and quality remain a challenge. With transient transfection processes established as a manufacturing platform for multiple commercial AAV products, there remains significant yield, cost, robustness, and scalability constraints that need to be resolved to enable a reliable supply of rAAV products for global patient access. The development of stable producer cell lines for rAAV products has enabled scalability and, in some cases, improvements in productivity. Herein we describe a novel AAV perfusion-enhanced expression (APEX) process, resulting in higher maximum cell densities in the production bioreactor with a 3- to 6-fold increase in volumetric productivity. This process has been successfully demonstrated across multiple serotypes in large scale cell culture with titers approaching 1×1012 GC/mL. The APEX production platform marks a significant leap forward in the efficient and effective manufacturing of rAAV vector products.

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.

Evaluation of Cellular Responses by Chlamydomonas reinhardtii in Media Containing Dairy-Processing Residues Derived from Cheese as Nutrients by Analyzing Cell Growth Activity and Comprehensive Gene Transcription Levels.

Utilities of whey powder (WP) and whey protein concentrate 34% powder (WPC34) prepared as dairy-processing residues were evaluated using a green alga Chlamydomonas reinhardtii. Analysis of C. reinhardtii growth showed that the strain used WP and WPC34 as nitrogen sources. Its specific growth rate and maximum cell density in WP-containing medium were higher than those in WPC34-containing medium; growth with WPC34 was improved by adding KCl or K2HPO4, which content was decreased as a result of WPC34's preparation from WP. Although the lipid contents in media containing dairy-processing residues were 2.72 ± 0.31 wt% and 2.62 ± 0.20 wt% with no significant difference, the composition ratio of fatty acid C14 with WPC34 was higher than that with WP and the composition ratio of the sum of fatty acid-C16 and -C18 with WPC34 tended to be lower than that with WP. Additionally, analyses of gene transcription showed that the transcription level of acetyl-CoA carboxylase biotin carboxyl carrier protein in WPC34-containing medium was lower than that in WP-containing medium, possibly affecting the ratios of the chain lengths of fatty acids. The transcription of genes involved in glycolysis and the TCA cycle was outstandingly lower in algae grown in WPC34-containing medium when compared to those cultivated in the presence of WP, resulting in differences in energy production for cell proliferation.

Optimization of Spore Production in Bacillus coagulans Using Response Surface Methodology Approach.

Due to its spore-forming ability, Bacillus coagulans has advantages over the other non-spore-forming probiotics. Among them, survival and stability during food processing and storage, resistance to acid pH, and digestive enzymes are important. However, there are few studies on the quality and amount of sporulation in B. coagulans. This study investigated the spore densities and formation efficiency of B. coagulans. The optimal medium formulation consisted of yeast extract (1.00g L-1), potassium acetate (20.00g L-1), and MnSO4 (0.01g L-1 and 0.03g L-1). After reaching the optimal medium, a response surface regression equation was established based on the results of central composite design (CCD) experimental designs to optimize time, temperature, and pH parameters. The predicted results thus obtained were in good agreement (R2 = 95.19%) with the results obtained by performing experiments. Multiple regression analysis and analysis of variance (ANOVA) showed that pH is negative, and temperature and time dose are positive factors. The maximum spore cell densities by optimization plots have obtained 9.80 log at temperature 83.77°C, pH 3.05, and time 111.19h, considering that B. coagulans needs special environmental and cellular conditions to enter the sporulation stage. In this study, the composition of the culture medium and factors such as temperature, time, and pH were considered influencing factors in B. coagulans sporulation.

In vitro competition between two transmissible cancers and potential implications for their host, the Tasmanian devil.

Since the emergence of a transmissible cancer, devil facial tumour disease (DFT1), in the 1980s, wild Tasmanian devil populations have been in decline. In 2016, a second, independently evolved transmissible cancer (DFT2) was discovered raising concerns for survival of the host species. Here, we applied experimental and modelling frameworks to examine competition dynamics between the two transmissible cancers invitro. Using representative cell lines for DFT1 and DFT2, we have found that in monoculture, DFT2 grows twice as fast as DFT1 but reaches lower maximum cell densities. Using co-cultures, we demonstrate that DFT2 outcompetes DFT1: the number of DFT1 cells decreasing over time, never reaching exponential growth. This phenomenon could not be replicated when cells were grown separated by a semi-permeable membrane, consistent with exertion of mechanical stress on DFT1 cells by DFT2. A logistic model and a Lotka-Volterra competition model were used to interrogate monoculture and co-culture growth curves, respectively, suggesting DFT2 is a better competitor than DFT1, but also showing that competition outcomes might depend on the initial number of cells, at least in the laboratory. We provide theories how the invitro results could be translated to observations in the wild and propose that these results may indicate that although DFT2 is currently in a smaller geographic area than DFT1, it could have the potential to outcompete DFT1. Furthermore, we provide a framework for improving the parameterization of epidemiological models applied to these cancer lineages, which will inform future disease management.

Antagonistic activity of Phaeobacter piscinae against the emerging fish pathogen Vibrio crassostreae in aquaculture feed algae.

Aquaculture provides a rich resource of high-quality protein; however, the production is challenged by emerging pathogens such as Vibrio crassostreae. While probiotic bacteria have been proposed as a sustainable solution to reduce pathogen load in aquaculture, their application requires a comprehensive assessment across the aquaculture food chain. The purpose of this study was to determine the antagonistic effect of the potential probiotic bacterium Phaeobacter piscinae against the emerging fish pathogen V. crassostreae in aquaculture feed algae that can be an entry point for pathogens in fish and shellfish aquaculture. P. piscinae strain S26 produces the antibacterial compound tropodithietic acid (TDA). In a plate-based assay, P. piscinae S26 was equally to more effective than the well-studied Phaeobacter inhibens DSM17395 in its inhibition of the fish pathogens Vibrio anguillarum 90-11-286 and V. crassostreae DMC-1. When co-cultured with the microalgae Tetraselmis suecica and Isochrysis galbana, P. piscinae S26 reduced the maximum cell density of V. crassostreae DMC-1 by 2 log and 3-4 log fold, respectively. A TDA-deficient mutant of P. piscinae S26 inhibited V. crassostreae DMC-1 to a lesser extent than the wild type, suggesting that the antagonistic effect involves TDA and other factors. TDA is the prime antagonistic agent of the inhibition of V. anguillarum 90-11-286. Comparative genomics of V. anguillarum 90-11-286 and V. crassostreae DMC-1 revealed that V. crassostreae DMC-1 carries a greater arsenal of antibiotic resistance genes potentially contributing to the reduced effect of TDA. In conclusion, P. piscinae S26 is a promising new candidate for inhibition of emerging pathogens such as V. crassostreae DMC-1 in algal feed systems and could contribute to a more sustainable aquaculture industry.IMPORTANCEThe globally important production of fish and shellfish in aquaculture is challenged by disease outbreaks caused by pathogens such as Vibrio crassostreae. These outbreaks not only lead to substantial economic loss and environmental damage, but treatment with antibiotics can also lead to antibiotic resistance affecting human health. Here, we evaluated the potential of probiotic bacteria, specifically the newly identified strain Phaeobacter piscinae S26, to counteract these threats in a sustainable manner. Through a systematic assessment of the antagonistic effect of P. piscinae S26 against V. crassostreae DMC-1, particularly within the context of algal feed systems, the study demonstrates the effectiveness of P. piscinae S26 as probiotic and thereby provides a strategic pathway for addressing disease outbreaks in aquaculture. This finding has the potential of significantly contributing to the long-term stability of the industry, highlighting the potential of probiotics as an efficient and environmentally conscious approach to safeguarding aquaculture productivity against the adverse impact of pathogens.

Effects of low concentration of gallic acid on the growth and microcystin production of Microcystis aeruginosa

Gallic acid (GA) is an allelochemical that has been utilized in high concentrations for the management of harmful algal blooms (HABs). However, there is limited knowledge regarding its impact on the growth of M. aeruginosa as the GA concentration transitions from high to low during the HABs control process. This study has revealed that as the GA concentration decreases (from 10 mg/L to 0.001 μg/L), a dose-response relationship becomes apparent in the growth of M. aeruginosa and microcystin production, characterized by high-dose inhibition and low-dose stimulation. Notably, at the concentration of 0.1 μg/L GA, the most significant growth-promoting effect on both growth and MCs synthesis was observed. The growth rate and maximum cell density were increased by 1.09 and 1.16 times, respectively, compared to those of the control group. Additionally, the contents of MCs synthesis saw a remarkable increase, up by 1.85 times. Furthermore, lower GA concentrations stimulated the viability of cyanobacterial cells, resulting in substantially higher levels of reactive oxygen species (ROS) and chlorophyll-a (Chl a) compared to other concentrations. Most importantly, the expression of genes governing MCs synthesis was significantly upregulated, which appears to be the primary driver behind the significantly higher MCs levels compared to other conditions. The ecological risk quotient (RQ) value of 0.1 μg/L GA was the highest of all experimental groups, which was approximately 30 times higher than that of the control, indicating moderate risk. Therefore, it is essential to pay attention to the effect of M. aeruginosa growth, metabolism and water ecological risk under the process of reducing GA concentration after dosing during the HABs control process.

Heavy toxic sludge tolerance in microalgae: Effect of energy generation metabolisms from C. humicola under IAA applications

Industrial sludge is characterized by high moisture content, complex composition, and significant organic toxicity. This research aimed to improve the performance of Chlorococcum humicola in removing hydroquinone-domesticated sludge toxicity through the addition of indole-3-acetic acid (IAA). The maximum cell density and dry weight of C. humicola were attained when the IAA concentration was 5.0 × 10−6 mol/L, at 6.79 × 107 cells/mL and 2.60 g/L, respectively. The toxicity of the sludge degradation rate reached its peak level of 59.70% on the 20th day. The toxicity reduction was consistent with the decrease of RNA degradation-related proteins such as A5FN12, which can be hydrolyzed and absorbed by C. humicola via active transport. In the presence of IAA, C. humicola increased its energy production processes, such as glycolysis and oxidative phosphorylation, to facilitate active transport. Moreover, the efficiency of signal transduction was improved, leading to enhanced cellular metabolism. The suppression of biosynthesis of glutathione and arginine at the molecular level may contribute to this observed trend. Overall, this study provides new insights into the mechanism by which IAA promotes the degradation of sludge toxicity by C. humicola, offering a novel method and theoretical basis for the safe and resource-based utilization of industrial sludge.

The influence of the environment on the diversity of Euglena species and their abundance in the lagoon of Nador-Morocco

The main objective of this work is to study the spatial and temporal evolution and distribution of Euglena species in the Nador lagoon. The study is based on four sampling stations and covers two specific seasons, namely spring and summer 2018. Euglenes belonging to the genus Euglena are particularly interesting and well known in the field of research, due to their great diversity in terms of pigmentation, size and morphological characteristics. Samples taken at each of the four sampling stations were carefully identified morphologically using an inverted light microscope. In total, five species belonging to the genus Euglena were identified, namely: Euglena viridis O.F. Müller 1786, Euglena Caudata Hübner 1886, Euglena Proxima P.A. Dangeard 1902, Euglena tuberculata Svirenko 1915, Euglena sp. Quantitative analysis of the species collected reveals some interesting results. The maximum cell density was recorded at station 4, located near the wastewater treatment plant, with a value of 55 cells per litre during the summer of 2019. In contrast, the minimum cell density was recorded at the same station 4, corresponding to Kariat Arekmen, with a value of 5 cells per litre during the spring period of 2019. These observations highlight significant variations in Euglenes cell density depending on geographical location and season.

The efficient capture of polysaccharides in Tetradesmus obliquus of indole-3-acetic acid coupling sludge extraction

Polysaccharide is an important biomass of algae. The sludge extract is rich in organic substances, which can be used by algae for biomass growth and high-value biomass synthesis, but its organic toxicity has an inhibitory effect on algae. To overcome inhibition and improve polysaccharide enrichment, Tetradesmus obliquus was cultured with sludge extract with different indole-3-acetic acid (IAA) concentrations. Within 30 days of the culture cycle, T. obliquus showed in good condition at the IAA dosage content of 10−6 M, the maximum cell density and dry weight were respectively (106.78 ± 2.20) × 106 cell/mL and 2.941 ± 0.067 g/L while the contents of chlorophyll-a, chlorophyll-b, and carotenoid were 1.79, 1.91 and 2.80 times that of the blank group, respectively. The highest polysaccharide accumulation was obtained under this culture condition, reaching 533.15 ± 21.11 mg/L on the 30th day, which was 2.49 times that in the blank group. By FT-IR and NMR analysis, it was found that the polysaccharides of T. obliquus were sulfated polysaccharide with glucose and rhamnose as the main monosaccharides. Proteomic showed that the up-regulation of A0A383WL26 and A0A383WLM8 enhanced the light trapping ability, and A0A383WMJ2 enhanced the accumulation of NADPH. The up-regulation of A0A383WHD5 and A0A383WAY6 indicated that IAA culture could repair the damage caused by sludge toxicity, thus promoting the accumulation of biomass. The above findings provided new insights into the mechanism of sludge toxicity removal of T. obliquus and the enhancement of the polysaccharide accumulation effect under different concentrations of IAA.

Remediation of aniline-contaminated aquifer by combining in-well Rhizobium borbori and circulated groundwater electrolysis

Aniline has become a common groundwater contaminant due to its wide use as a raw material in agriculture and pharmaceutical products. The current technologies for in situ remediation of aniline in groundwater are limited by the strains deficient in bacterial species, limited oxygen supply, excessive waste gas load and cost. Accordingly, we conducted a laboratory sand tank experiment to remediate groundwater contaminated with aniline by combining circulated groundwater electrolysis and in-well Rhizobium borbori, which was isolated from activated sludge. The results of the experiment indicated that the optimum concentration of aniline for Rhizobium borbori is about 5 mg/L, beyond which the maximum cell density and the highest specific growth rate decreases as the aniline concentration increases. The optimized duration for immobilizing the Rhizobium borbori into the bioreactor is 4–5 days. Though the Rhizobium borbori was strongly inhibited by the high-concentration of aniline, the immobilized bioreactor in the 350 mg/L aniline solution successfully formed biofilm. The aniline volatilization had limited influence on the observation of bioremediation performance, and the combination of circulated groundwater and in-well Rhizobium borbori supplied a steady dose of oxygen to the bioreactor efficiently degrading the entire region between the injection and extraction well. In addition, a numerical model for the sand tank remediation experiment was used to estimate the yield coefficient of oxygen to be 0.484 g/g, which indicates the presence of ammonia nitrogen as by-products; accordingly, a smaller wellbore size as well a higher circulation flow rate and intensity of current are recommended to improve the water quality. Despite the positive outcomes and potential of the newly developed technology to degrade subsurface aniline, parallel experiments should be conducted to estimate the environmental risk of the by-products and explore the controlling mechanisms of each component in this comprehensive system.

Optimisation of lutein production potential in marine biflagellate Proteomonas sulcata isolated from Indian coastal waters

The present study was conducted to isolate, identify, and extract the lutein from a biflagellate Proteomonas sulcata from the coastal waters of the Gulf of Mannar, Southern east coast of India. The targeted unicellular biflagellate P. sulcata belonging to the group Geminigeraceae was collected and isolated using 96 well plates followed by the serial dilution method and morphologically confirmed. However, the pure isolate was also confirmed based on molecular identification by PCR and sequence analysis of 18 S rRNA. The resultant sequence was analysed and reflected in phylogenetic tree. Optimised the environmental parameters to obtain the maximum cell density of 50.36 ± 0.81 × 105 cells/ml. The unique characteristic feature of the isolate was the change in colour during the culture phases. ie., red colour in growing phase up to 6th day in haffkine flask and changed into green during the stationary phase until the death phase. The reason for the colour change might be due to phycobiliprotein present in the microalga and also confirmed by FTIR analysis. The maximum chlorophyll content and carotenoid of 4.59 ± 0.38 mg/g 2.55 ± 0.12 mg/g was estimated. The conventional lutein extraction process such as saponification and solvent extraction methods was followed. The process was optimized and extracted crude lutein was analysed by using HPLC. The present study concluded that, the maximum lutein yield of 3.96 ± 0.1 mg/g was obtained at a 6 M concentration of KOH and also found that the highest amount of lutein was obtained from wet biomass (red-coloured biomass) of 4.72 ± 0.38 mg/g which is significantly higher than that of freeze-dried biomass.