Culture Density Research Articles

Unbiased identification of cell identity in dense mixed neural cultures.

Induced pluripotent stem cell (iPSC) technology is revolutionizing cell biology. However, the variability between individual iPSC lines and the lack of efficient technology to comprehensively characterize iPSC-derived cell types hinder its adoption in routine preclinical screening settings. To facilitate the validation of iPSC-derived cell culture composition, we have implemented an imaging assay based on cell painting and convolutional neural networks to recognize cell types in dense and mixed cultures with high fidelity. We have benchmarked our approach using pure and mixed cultures of neuroblastoma and astrocytoma cell lines and attained a classification accuracy above 96%. Through iterative data erosion, we found that inputs containing the nuclear region of interest and its close environment, allow achieving equally high classification accuracy as inputs containing the whole cell for semi-confluent cultures and preserved prediction accuracy even in very dense cultures. We then applied this regionally restricted cell profiling approach to evaluate the differentiation status of iPSC-derived neural cultures, by determining the ratio of postmitotic neurons and neural progenitors. We found that the cell-based prediction significantly outperformed an approach in which the population-level time in culture was used as a classification criterion (96% vs 86%, respectively). In mixed iPSC-derived neuronal cultures, microglia could be unequivocally discriminated from neurons, regardless of their reactivity state, and a tiered strategy allowed for further distinguishing activated from non-activated cell states, albeit with lower accuracy. Thus, morphological single-cell profiling provides a means to quantify cell composition in complex mixed neural cultures and holds promise for use in the quality control of iPSC-derived cell culture models.

Multiplex genome editing eliminates lactate production without impacting growth rate in mammalian cells.

The Warburg effect, which describes the fermentation of glucose to lactate even in the presence of oxygen, is ubiquitous in proliferative mammalian cells, including cancer cells, but poses challenges for biopharmaceutical production as lactate accumulation inhibits cell growth and protein production. Previous efforts to eliminate lactate production in cells for bioprocessing have failed as lactate dehydrogenase is essential for cell growth. Here, we effectively eliminate lactate production in Chinese hamster ovary and in the human embryonic kidney cell line HEK293 by simultaneous knockout of lactate dehydrogenases and pyruvate dehydrogenase kinases, thereby removing a negative feedback loop that typically inhibits pyruvate conversion to acetyl-CoA. These cells, which we refer to as Warburg-null cells, maintain wild-type growth rates while producing negligible lactate, show a compensatory increase in oxygen consumption, near total reliance on oxidative metabolism, and higher cell densities in fed-batch cell culture. Warburg-null cells remain amenable for production of diverse biotherapeutic proteins, reaching industrially relevant titres and maintaining product glycosylation. The ability to eliminate lactate production may be useful for biotherapeutic production and provides a tool for investigating a common metabolic phenomenon.

Effect of Different Stocking Densities on Growth, Survival and Blood Parameters of Pacific Fat Sleeper Dormitator latifrons in a Small-Scale Aquaponic System

Aquaponics is the integration between aquaculture and hydroponics, where bacteria mediate the interaction between fish and plants, facilitating a mutually beneficial system. Although there have been numerous studies on aquaponics, there have been few studies focused on optimizing management parameters in small-scale systems for family farming. A key aspect is to find the appropriate culture density to maximize the production of both fish and plants. In aquaculture, introduced species have predominated due to their ease of management, great adaptability, and high growth rates; however, it is important to consider alternatives such as Dormitator latifrons. To determine the effect of density on the yield of D. latifrons and chard Beta vulgaris in an aquaponic culture, three culture densities of 10, 30, and 50 fish m−3 were evaluated in triplicate with a fixed plant density of 20 plants m−2. Nine experimental units (EU) were implemented consisting of 300 L Rotoplas® troughs, a settler (80 L), a biofilter (80 L), and a submersible water pump connected to a nutrient film technique (NFT) system that represented the hydroponic component. The results obtained suggest that an intermediate stocking density (30 fish m−3) could provide a favorable balance between fish and plant performance and fish health. These findings contribute to the knowledge on the culture of this species in aquaponics.

Bacterial microbiota associated with raw plant-based meat analogue products and their influences on selective enrichment for Escherichia coli O157:H7

Towards fostering a more sustainable food production system in face of the climate change challenge, alternative protein meat-substitute products that are plant-based and free of animal by-products have been gaining attractions from both food manufacturers and consumers. With these so-called plant-based meat analogues (PBMAs) becoming increasingly available at supermarkets, there is very little known about their microbial properties. In this short report, we characterized the bacterial composition of raw plant-based ground meat imitation retail products using 16S rRNA gene amplicon sequencing. Despite the observed bacterial community dissimilarity between sample brands, a total of 18 shared genera (dominated by Bacilli and Gammaproteobacteria classes) were identified as the core constituents of the bacterial microbiota of these PBMA products. Within the scope of food safety testing, to gain insights on the dynamics of the enrichment process for E. coli O157:H7 in accordance with the Health Canada reference method MFHPB-10, bacterial taxonomic analyses were conducted at different stages of the prescribed cultural procedures. Using both control and E. coli O157:H7-inoculated PBMA samples it was revealed that, independent of the presence of E. coli O157:H7, off-target bacteria of the Clostridium sensu stricto 1 genus were significantly enriched from the uncultured samples. Additionally, the abundance of Hafnia-Obesumbacterium bacteria in the PBMA samples was also increased in the enrichment products, but only when E. coli O157:H7 was absent. Consistent with the spread-plating results indicating that the inoculated E. coli O157:H7 cells were capable of reaching a high density (>108 CFU/ml) in the resultant enrichment cultures, the significant enrichment of bacterial 16S rRNA gene sequences belonging to the targeted genus of Escherichia, but not Hafnia-Obesumbacterium. This further highlights the competitive nature of the selective enrichment for E. coli O157:H7 against specific background bacteria associated with the PBMA products.

Gain of 1q confers an MDM4-driven growth advantage to undifferentiated and differentiating hESC while altering their differentiation capacity

Gain of 1q is a highly recurrent chromosomal abnormality in human pluripotent stem cells. In this work, we show that gains of 1q impact the differentiation capacity to derivates of the three germ layers, leading to mis-specification to cranial placode and non-neural ectoderm during neuroectoderm differentiation. Also, we found a weaker expression of lineage-specific markers in hepatoblasts and cardiac progenitors. Competition assays show that the cells retain their selective advantage during differentiation, which is mediated by a higher expression of MDM4, a gene located in the common region of gain. MDM4 drives the winner phenotype of the mutant cells in both the undifferentiated and differentiating state by reducing the cells’ sensitivity to DNA damage through decreased p53-mediated apoptosis. Finally, we found that cell density in culture plays a key role in promoting the competitive advantage of the cells by increasing DNA damage.

Red Light Responsive Cre Recombinase for Bacterial Optogenetics.

Optogenetic tools have been used in a wide range of microbial engineering applications that benefit from the tunable, spatiotemporal control that light affords. However, the majority of current optogenetic constructs for bacteria respond to blue light, limiting the potential for multichromatic control. In addition, other wavelengths offer potential benefits over blue light, including improved penetration of dense cultures and reduced potential for toxicity. In this study, we introduce OptoCre-REDMAP, a red light inducible Cre recombinase system in Escherichia coli. This system harnesses the plant photoreceptors PhyA and FHY1 and a split version of Cre recombinase to achieve precise control over gene expression and DNA excision. We optimized the design by modifying the start codon of Cre and characterized the impact of different levels of induction to find conditions that produced minimal basal expression in the dark and induced full activation within 4 h of red light exposure. We characterized the system's sensitivity to ambient light, red light intensity, and exposure time, finding OptoCre-REDMAP to be reliable and flexible across a range of conditions. In coculture experiments with OptoCre-REDMAP and the blue light responsive OptoCre-VVD, we found that the systems responded orthogonally to red and blue light inputs. Direct comparisons between red and blue light induction with OptoCre-REDMAP and OptoCre-VVD demonstrated the superior penetration properties of red light. OptoCre-REDMAP's robust and selective response to red light makes it suitable for advanced synthetic biology applications, particularly those requiring precise multichromatic control.

Applying Beer's Law in the undergraduate cell biology laboratory: examining the mathematical relationship between optical density, cell concentration, and cell size using budding yeast.

Undergraduate students majoring in the life sciences benefit from experience with data analyses that connect mathematical calculations to the biological systems they are studying. Monitoring the optical density and cell number of Saccharomyces cerevisiae liquid cultures allows students to gain quantitative experience generating standard curves and trendlines that capture the relationship between optical density and cell concentration for a given S. cerevisiae strain. Data comparisons across multiple strains can yield insights into the biophysical properties of cells that drive light absorbance and scattering. In this Tips and Tools article, we share a laboratory module that allows students to experience cell biology tools, laboratory measurements, and data analysis to determine the mathematical relationship between optical density and cell concentration in liquid microbial cultures. This module could be integrated into undergraduate classes ranging from general biology to upper-level cell biology or microbiology and can be a starting point for more complex investigations of microbial growth.

Biomass Productivity and Photosynthetic Activity in Ulva compressa (Chlorophyta) in Raceway Photobioreactors Under Stress Conditions.

Research in seaweed cultivation technologies aims to increase production and reduce costs, leading to more efficient and sustainable processes. In this study, we analyzed the outdoor production of Ulva compressa cultured in summertime at different stocking densities of 0.6, 0.8 and 1.0 kg Fresh weight (FW) m-2 in a raceway photobioreactor with 30 m2 surface (3000 L), and its relation to photosynthetic activity. Under the experimental conditions of high temperature (>28-30 °C) and pH > 9 in culture water, higher seaweed density resulted in lower specific growth rate. The biomass production has been related to photosynthetic activity by using in vivo chlorophyll a fluorescence. Dynamic photoinhibition was observed at noon, which was less severe in cultures with higher algal densities. However, photosynthesis recovered in the afternoon. Seaweeds that were acclimatized for a week to the conditions of 1.0 kg FW m-2 stocking density showed an increase in biomass growth and absence of photoinhibition compared to non-acclimatized thalli. In conclusion, the cultivation of U. compressa in a mid-scale raceway photobiorreactor under conditions of high irradiance and temperature and low nutrient input, exhibited the best photosynthetic performance and hence the highest growth rates for the highest culture density assayed (1.0 kg FW m-2).

Optimization of manufacturing process for serotype 14 pneumococcal capsular polysaccharide

Streptococcus pneumoniae is a pathogenic bacterium that causes infections such as pneumonia, meningitis, otitis media, and bacteremia. The prevention of pneumococcal disease by vaccination has become more urgent due to increased antibiotic resistance. Pneumococcal capsular polysaccharides (CPS) are effective vaccine antigens that stimulate the host to produce protective antibodies. S. pneumoniae serotype 14 is one of most prevalent types in Latin America and across the world. However, the yield of S. pneumoniae serotype 14 CPS from existing fermentation processes remains low and requires improvement. In this study, various aspects of the fermentation process were optimized to improve pneumococcal growth and polysaccharide productivity, including feed medium, cultivation gas environment, fermentation pH, and temperature. A simplified purification method was also developed to obtain pure CPS, including ultrafiltration, acid and ethanol precipitation, diafiltration, and lyophilization. These fermentation optimizations significantly enhanced the optical density of pneumococcal bacterial cultures and increased fermentation yields to 2.4–2.6 g/L—significantly higher than previously achieved. Furthermore, the test results of pure CPS could meet the requirements in the European Pharmacopoeia (11th edition). These optimizations provide valuable insights into the nutritional requirements and impact of varying fermentation process parameters on pneumococcal growth and CPS productivity, thus contributing to the development of a more efficient and cost-effective method for the production of pneumococcal CPS—essential for manufacturing vaccines against pneumococcal infections.

Micro RNA-175 Targets Claudin-1 to Inhibit Madin–Darby Canine Kidney Cell Adhesion

Background: The Madin–Darby canine kidney (MDCK) cell line constitutes a key component of influenza vaccine production, but its dependence on adherent growth limits cell culture density and hinders vaccine yield. There is evidence that the use of gene editing techniques to inhibit cell adhesion and establish an easily suspended cell line can improve vaccine yield; however, the mechanisms underlying MDCK cell adhesion are unclear. Methods: In this study, we used transcriptomics to analyse differentially expressed mRNAs and miRNAs in adherent and suspension cultures of MDCK cells. Results: We found that claudin-1 (CLDN1) expression was downregulated in the suspension MDCK cells and that CLDN1 promotes MDCK cell–extracellular matrix adhesion. Additionally, microRNA (miR)-175 expression was upregulated in the suspension MDCK cells. Importantly, we demonstrated that miR-175 inhibits MDCK cell adhesion by targeting the CLDN1 3′-untranslated region (UTR). These findings contribute to a more comprehensive understanding of the regulatory mechanisms modulating cell adhesion and provide a basis for establishing suspension-adapted, genetically engineered cell lines. Our work could also facilitate the identification of targets for tumour therapy.

Comparison of Decontamination-Preservation Protocols for Delayed Surgical Re-implantation of Osteoarticular Fracture Fragments

Background: To avoid discarding contaminated, devascularized osteoarticular fragments required for joint reconstruction, fragments need to be decontaminated while preserving chondrocyte viability. We hypothesized that disinfection with povidone-iodine or chlorhexidine followed by preservation using the Missouri Osteochondral Preservation System (MOPS) would allow for effective decontamination while retaining essential chondrocyte viability in osteoarticular fracture fragments for up to 14 days of shelf-stable point-of-care storage. Methods: With IACUC approval, purpose-bred hounds (n=16) were humanely euthanized for unrelated purposes and subjected to captive bolt trauma to create open distal humeral fractures. For each elbow (n=32), humerus, radius and ulna tissues were recovered such that 96 contaminated, devascularized osteoarticular fragments were randomly allocated to one treatment: Betadine (n=42): saline irrigation (1L), immersion in 10% povidone-iodine (20 min), saline irrigation; Chlorhexidine (n=42): saline irrigation, immersion in 0.002% chlorhexidine gluconate (20 min), saline irrigation; Injured Control (n=12): no decontamination treatment. After 7 or 14 days in MOPS, tissues were assessed by quantitative microbial culture and Viable Chondrocyte Density (VCD) measures. Results: Captive bolt trauma consistently resulted in type 3 open articular fractures. Injured Control osteoarticular fragments produced high polymicrobial counts at days 7 and 14. Chlorhexidine treatment was effective for decontaminating fragments such that no CFUs for clinically relevant bacteria were produced, while Betadine treatment was not fully effective at decontamination. Chlorhexidine decontamination followed by MOPS preservation maintained VCD in osteoarticular tissues over the desired 70% mean for 14 days, whereas the Injured Control group was associated with significant loss of VCD (Day-7=59%, Day-14=13%), which was further exacerbated by Betadine treatment (Day-7=29%, Day-14=6%). Conclusion: Contaminated, devascularized osteoarticular fracture fragments can be effectively decontaminated while maintaining essential chondrocyte viability for 14 days after type 3 open articular fractures using a decontamination-preservation protocol that combines saline irrigation with 0.002% chlorhexidine immersion followed by shelf-stable point-of-care storage in MOPS.

B-225 Bridging the Gap Between Clinical Microbiology and Molecular Diagnostics: Correlation of PCR Cycle Threshold Values for Establishing Semi-Quantitative CFU/mL Ranges of Bacterial Targets

Abstract Background Molecular diagnostics (MolDx) has surpassed culture in the clinical microbiology laboratory. Wong (2023) states PCR is more sensitive than urine culture in detecting polymicrobial infections (para. 8), marking an advantage in MolDX for urinary tract infections. This advancement poses a challenge for clinicians in treatment decision-making. To address requests for numerical MolDx result ranges, a validation accessed semi-quantitative Colony Forming Units per milliliter (CFU/mL) through the assessment of PCR cycle threshold (CT) values. Methods Bacterial targets of UTI and STI PCR panels were inoculated in nutrient broth (Hardy diagnostics cat#K43) and incubated overnight in a shaking incubator at 35±2°C. The density of the cultures was measured using the OD600 function of a NanoDrop One/One after 18-24 hour incubation. The density of stock cultures was adjusted to 106 CFU/mL. Serial dilutions of stock were made in 1:10 steps covering the range of 106 to 100 CFU/mL. The stock and dilutions were extracted, and PCR was performed in a minimum of 2 replicates of each sample for the UTI and STI PCR panels. Mean CT values, standard deviations and coefficients of variation were calculated for each sample. The linearity of CT values within each organism’s stock and dilution samples were assessed by evaluating the CT differences between each dilution step. Corresponding CFU/mL concentrations were tabulated against each mean CT value. Results The average CT values for reportable CFU/mL ranges were established for each stock and dilution. Mean CT values for each CFU/mL breakdown across all organisms were also calculated, along with standard deviations, to investigate universal CT-CFU/mL correlations. A final interpretation table (Table 1) was constructed according to the universal range calculations. Conclusions The study confirmed correlation between CT values and CFU/mL ranges. Further data analysis and comparison with other laboratories’ results will enhance accuracy and precision of reportable CFU/mL ranges.

Improving trans‐cinnamic acid production in a model cyanobacterium

Abstracttrans‐Cinnamic acid (tCA) is a precursor in the synthesis of many high‐value compounds with bio‐active qualities useful in applications like medicine, polymers, and cosmetics. Currently tCA is produced by industrial chemical synthesis from fossil fuels or cost‐prohibitive isolation from terrestrial plants. Cyanobacteria, a type of photosynthetic bacteria, can be readily engineered to convert sunlight and carbon dioxide into metabolites of interest at relatively high amounts compared to terrestrial plants. The purpose of this study is to advance the industrial and commercial value of cyanobacteria as a biological factory for renewable production of tCA. Production of tCA has previously been demonstrated in the model cyanobacterium Synechocystis sp. PCC 6803 (S. 6803) via expression of non‐native phenylalanine ammonia lyase (PAL) from various organisms. This project focuses on developing and characterizing a new high‐titer strain of S. 6803 expressing a plant PAL gene controlled by an inducible promoter. We assessed production in shake flasks under constant light, a 12 h:12 h light:dark cycle, and environmental photobioreactors (ePBRs) with a sinusoidal, rapidly fluctuating light environment. Our strain demonstrates a four‐fold increase in tCA production to ~500 mg L−1 by 14 days compared to previously reported titers in S. 6803 under shake flask cultivation and a 30–50% improved average tCA production per culture density (60 mg·L−1·OD730−1) in ePBRs over comparable previously reported culture methods. Our study progresses S. 6803 tCA bioproduction into higher culture volumes, up to 500 mL, while further validating the strength of an inducible system for tCA production in S. 6803.

High Precision Ping-Pong Auto-Zeroed Lock-in Fluorescence Photometry Sensor.

This paper presents a high-precision CMOS fluorescence photometry sensor using a novel lock-in amplification scheme based on switched-biasing and ping-pong auto-zeroing techniques. The CMOS sensor includes two photodiodes and a lock-in amplifier (LIA) operating at 1 kHz. The LIA comprises a differential low-noise amplifier using a novel switched-biasing ping-pong auto-zeroed scheme, an automatic phase aligner, a programmable gain amplifier, a band-pass filter, a mixer, and an output low-pass filter. The design is fabricated in 0.18-µm CMOS process, and the measurement shows that the LIA can retrieve noisy input signals with a dynamic reserve of 42 dB, while consuming only 0.7 mW from a 1.8 V supply voltage. The measured results show that the LIA can detect a wide range of incident light power from 8 nW to 24 µW. The proposed design is encapsulated in a 3D-printed housing allowing for real-time invitro biomarker detection. This ambulatory platform uses an LED and a fiber optic to convey the excitation light to the sample and retrieve the fluorescence signal. Experiments with a beads solution diluted in PBS demonstrate that the sensor has a sensitivity of 1:100 k. Experimental results obtained invitro with NIH3T3 mouse cells tagged with membrane dye show the ability of the prototype to detect different densities of cell culture. The portable prototype, which includes optical filters and a small 30 mm × 36 mm × 30 mm printed circuit board enclosed inside the 3D-printed housing, consumes 36.7 mW and weighs 120 g.

Expression pattern of the fused in sarcoma gene and its contextual influence on the density-specific response of the growth hormone/insulin-like growth factor 1 axis in zig-zag eels (Mastacembelus armatus)

The fused in sarcoma (FUS) protein is a DNA/RNA binding protein from the ten-eleven translocation protein family that is associated with neurodegeneration, and it has been shown to promote cell proliferation through the growth hormone/insulin-like growth factor 1 (Gh/Igf-1) signaling pathway. The zig-zag eel (Mastacembelus armatus) is a newly discovered species exhibiting sexual dimorphism in growth, and the potential role of fus in the growth and development of this species remains largely unknown. Herein, we analyzed the homology, conserved domains, evolutionary characteristics, and conserved syntenies of fus in several teleost species. The expression of fus was predominant in the brain and exhibited sexual dimorphism in the brain, muscle, and liver of zig-zag eels. We found that microRNA (miR)-146-5p, miR-489-3p, and 24 other miRNAs were targeted to the fus 3′ untranslated region, which might affect muscle and bone development in adults. The igf1, insulin-like growth factor 1 receptor a (igf1ra), insulin-like growth factor 2 receptor (igf2r), growth hormone-releasing hormone-like receptor (ghrhrl), growth hormone secretagogue receptor type 1 (ghsr), and glucocorticoid receptor (gr) genes contained a higher abundance of GU-rich fus motifs compared to the other four genes analyzed in zig-zag eels. We also measured the expression of fus mRNA during fish culture at various stocking densities to further elucidate the relationship between fus expression and the Gh/Igf-1 axis. After 100 days of fish cultivation, the expression of fus and ghrhrl decreased and the expression of ghrh and gr increased as the culture density increased (p < 0.05). The expression of fus exhibited a remarkable positive correlation with a specific growth rate. These results indicate that fus mediates growth differences by regulating the expression of several growth-related genes including Gh/Igf-1 axis genes in zig-zag eels.

Computer-Simulated Virtual Image Datasets to Train Machine Learning Models for Non-Invasive Fish Detection in Recirculating Aquaculture.

Artificial Intelligence (AI) and Machine Learning (ML) can assist producers to better manage recirculating aquaculture systems (RASs). ML is a data-intensive process, and model performance primarily depends on the quality of training data. Relatively higher fish density and water turbidity in intensive RAS culture produce major challenges in acquiring high-quality underwater image data. Additionally, the manual image annotation involved in model training can be subjective, time-consuming, and labor-intensive. Therefore, the presented study aimed to simulate fish schooling behavior for RAS conditions and investigate the feasibility of using computer-simulated virtual images to train a robust fish detection model. Additionally, to expedite the model training and automate the virtual image annotation, a process flow was developed. The 'virtual model' performances were compared with models trained on real-world images and combinations of real and virtual images. The results of the study indicate that the virtual model trained solely with computer-simulated images could not perform satisfactorily (mAP = 62.8%, F1 score = 0.61) to detect fish in a real RAS environment; however, replacing a small number of the virtual images with real images in the training dataset significantly improved the model's performance. The M6 mixed model trained with 630 virtual and 70 real images (virtual-to-real image ratio: 90:10) achieved mAP and F1 scores of 91.8% and 0.87, respectively. Furthermore, the training time cost for the M6 model was seven times shorter than that for the 'real model'. Overall, the virtual simulation approach exhibited great promise in rapidly training a reliable fish detection model for RAS operations.

Multiplex genome editing eliminates the Warburg Effect without impacting growth rate in mammalian cells.

The Warburg effect is ubiquitous in proliferative mammalian cells, including cancer cells, but poses challenges for biopharmaceutical production, as lactate accumulation inhibits cell growth and protein production. Previous efforts to eliminate lactate production via knockout have failed in mammalian bioprocessing since lactate dehydrogenase has proven essential. However, here we eliminated the Warburg effect in Chinese hamster ovary (CHO) and HEK293 cells by simultaneously knocking out lactate dehydrogenase and regulators involved in a negative feedback loop that typically inhibits pyruvate conversion to acetyl-CoA. In contrast to long-standing assumptions about the role of aerobic glycolysis, Warburg-null cells maintain wildtype growth rate while producing negligible lactate. Further characterization of Warburg-null CHO cells showed a compensatory increase in oxygen consumption, a near total reliance on oxidative metabolism, and higher cell densities in fed-batch cell culture. These cells remained amenable for production of diverse biotherapeutic proteins, reaching industrially relevant titers and maintaining product glycosylation. Thus, the ability to eliminate the Warburg effect is an important development for biotherapeutic production and provides a tool for investigating a near-universal metabolic phenomenon.

Ala-Cys-Cys-Ala dipeptide dimer alleviates problematic cysteine and cystine levels in media formulations and enhances CHO cell growth and metabolism

Cysteine and cystine are essential amino acids present in mammalian cell cultures. While contributing to biomass synthesis, recombinant protein production, and antioxidant defense mechanisms, cysteine poses a major challenge in media formulations owing to its poor stability and oxidation to cystine, a cysteine dimer. Due to its poor solubility, cystine can cause precipitation of feed media, formation of undesired products, and consequently, reduce cysteine bioavailability. In this study, a highly soluble cysteine containing dipeptide dimer, Ala-Cys-Cys-Ala (ACCA), was evaluated as a suitable alternative to cysteine and cystine in CHO cell cultures. Replacing cysteine and cystine in basal medium with ACCA did not sustain cell growth. However, addition of ACCA at 4 mM and 8 mM to basal medium containing cysteine and cystine boosted cell growth up to 15% and 27% in CHO-GS and CHO–K1 batch cell cultures respectively and led to a proportionate increase in IgG titer. 13C-Metabolic flux analysis revealed that supplementation of ACCA reduced glycolytic fluxes by 20% leading to more efficient glucose metabolism in CHO–K1 cells. In fed-batch cultures, ACCA was able to replace cysteine and cystine in feed medium. Furthermore, supplementation of ACCA at high concentrations in basal medium eliminated the need for any cysteine equivalents in feed medium and increased cell densities and viabilities in fed-batch cultures without any significant impact on IgG charge variants. Taken together, this study demonstrates the potential of ACCA to improve CHO cell growth, productivity, and metabolism while also facilitating the formulation of cysteine- and cystine-free feed media. Such alternatives to cysteine and cystine will pave the way for enhanced biomanufacturing by increasing cell densities in culture and extending the storage of highly concentrated feed media as part of achieving intensified bioproduction processes.

Optimizing initial stocking densities of wild coral spawn slicks for mass production of larvae and settled corals for restoration

Mass culturing coral larvae collected from spawn slicks during spawning events and rearing them directly on reefs is an important method for coral reef restoration as it utilizes high genetic diversity and is readily upscaled. Understanding optimal initial stocking densities in mass cultures is, therefore, central to optimizing larval production efficiency. Yet, no studies have focused on optimizing initial stocking densities while coral embryos are still floating, despite the influence of density‐dependent effects during larval culturing. This study aimed to (1) explore initial stocking densities based on slick coverage and (2) determine the effects of initial densities on embryo development to coral settlement and early post‐settlement survival. Wild coral spawn slicks were collected at Lizard Island and reared at different combinations of slick coverage and density based on tank volume. Immediate density‐dependent effects were observed in treatments with 45% slick coverage, which had lower abundances of embryos 12 hours post‐spawning. The 12‐hour embryo densities ranged from 1.7 to 10.7 mL−1 and continued to display a negative relationship between density and survival of developing larvae. Overall, a density of approximately 5.1 embryos mL‐1 was the optimal stocking density for larval survival to competency (16.5%). The proportion of settlement and early post‐settlement survival was negatively correlated with higher initial densities, while a positive relationship was observed between the abundance of competent larvae and those settled, including initial settlers and 2‐month‐old recruits. These results highlight the importance of both larval quality and supply, and opportunities for site and system‐specific optimization of larval production in future work.

Mechanism Study on the Effect of Surface Electrical Property on Microbial Membrane Formation Efficiency of TiO2-SiC Composite Filler in Recirculating Aquaculture System.

Recirculating aquaculture systems (RASs) offer significant advantages in aquaculture by markedly decreasing water usage and increasing culture density. A vital component within a RAS is the filler material, which serves as a surface for microbial colonization. Effective microbial treatment is crucial for the efficient operation of a RAS as it assists in purifying the wastewater generated within the system. Nevertheless, traditional fillers often show low efficiency in biofilm formation. The commercial silicon carbide used in this study is a foam ceramic filter with a density of about 0.4-0.55 g/cm3, a number of holes of about 10, and a through porosity of 80.9%, with a diameter of about 5 cm. This research investigates the utilization of a titanium dioxide-silicon carbide (TiO2-SiC) composite filler to improve the purification efficiency of ammonia nitrogen and chemical oxygen demand (COD) in aquaculture wastewater. The study involved the application of titanium dioxide films onto the surface of silicon carbide to produce the composite filler. This method takes advantage of the dipole interaction between titanium dioxide and microorganisms, which enhances biofilm culturing efficiency on the silicon carbide surface. The performance of three different fillers was assessed for their ability to purify aquaculture wastewater. Results showed that the TiO2-SiC composite filler was 1.67 times more effective in removing COD and 1.07 times more effective in removing ammonia nitrogen compared to using silicon carbide alone. These results demonstrate that the incorporation of a titanium dioxide coating substantially boosts the microbial colonization efficiency of silicon carbide, thereby enhancing the overall wastewater purification efficiency in RAS.