Heat resistance of five spoilage microorganisms in a carbonated broth.

Despite their acidic pH, carbonated beverages can be contaminated by spoilage microorganisms. Thermal treatments, before and/or after carbonation, are usually applied to prevent the growth of these microorganisms. However, the impact of CO2 on the heat resistance of spoilage microorganisms has never been studied. A better understanding of the combined impact of CO2 and pH on the heat resistance of spoilage microorganisms commonly found in carbonated beverages might allow to optimize thermal treatment. Five microorganisms were selected for this study: Alicyclobacillus acidoterrestris (spores), Aspergillus niger (spores), Byssochlamys fulva (spores), Saccharomyces cerevisiae (vegetative cells), and Zygosaccharomyces parabailii (vegetative cells). A method was developed to assess the impact of heat treatments in carbonated media on microbial resistance. The heat resistances of the five studied species are coherent with the literature, when data were available. However, neither the dissolved CO2 concentration (from 0 to 7 g/L), nor the pH (from 2.8 to 4.1) have an impact on the heat resistance of the selected microorganisms, except for As. niger, for which the presence of dissolved CO2 reduced the heat resistance. This study improved our knowledge about the heat resistance of some spoilage microorganisms in presence of CO2.

The metabolic landscape of the bacteria Rhodococcus erythropolis used in the biodesulfurization of petroleum products: an emphasis on 2-hydroxybiphenyl.

Microorganisms produce diverse classes of metabolites under various physiological conditions. Many bacterial strains have been reported to carry out the process of desulfurization in a cost-effective manner by converting dibenzothiophene (DBT) into 2-hydroxybiphenyl (2-HBP) and then using the 2-HBP as a carbon source for growth and development. Key rate-limiting factors and an increased concentration of 2HBP (400 µM) affect the biodesulfurization activity of bacteria through the produced metabolites. Thus, this study was designed to explore the nature of the metabolites produced by Rhodococcus erythropolis in the presence of DBT and 2HBP supplemented with a culture medium. A total of 330 metabolites were detected, and the key metabolites identified were 11Z-eicosaenoyl-EA, 1-carboxyethylisoleucine, 1(3)-glyceryl-PGF2alpha, taurine, 2-hydroxynicotinic acid, 4,4-dimethyl-14alpha-hydroxymethyl-5alpha-cholest-8-en-3beta-ol, and 10-nitrooleic acid. The supplementation of DBT and DBT-2HBP resulted in the differential regulation of these metabolites, either through downregulation or overexpression. Furthermore, at high concentrations of 2-HBP, 1-carboxyethylisoleucine, taurine, 2-hydroxynicotinic acid, and nicotinic acid were upregulated. This work proposes that the identified metabolites may play a role in bacteria-mediated desulphurization and could be beneficial in developing a cost-effective method of desulphurization for refining petroleum.

Fermenter scale production of recombinant beta-mannanase by E. coli BL21 cells under microaerobic environment.

Aim of the study was to optimize and produce beta-mannanase at fermenter scale by using cheaper minimal media. Increased production of beta-mannanase from Microbacterium camelliasinensis CIAB417 was achieved by heterologous expression in E. coli BL21 (DE3). The scale-up production of beta-mannanase was optimized from shake flask to 5-L fermenter. The cost-effective minimal media (M9+e) without any vitamins was found to be most effective and optimized for culturing the cells. The same media displayed no significant fluctuation in the pH while culturing the cells for the production of beta-mannanase both at shake flask and fermenter level. Additionally, E. coli cells were able to produce similar amount of dry cell weight and recombinant beta-mannanase both in the presence of micro and macro-oxygen environment. The optimized media was demonstrated to show no significant drop in pH throughout the recombinant protein production process. In one litre medium, 2.0314 g dry weight of E. coli cells yielded 1.8 g of purified recombinant beta-mannanase. The purified enzyme was lyophilized and demonstrated to hydrolyse locust bean gum to release mannooligosaccharides.

Optimization of Hoagland solution macro-elements as a culture media, for increasing protein content of duckweeds (Lemna minor).

This study aimed to increase the protein content of duckweed, a promising alternative to animal proteins and a sustainable source of plant protein cultivated via soilless agriculture, by manipulating the culture medium conditions (Hoagland solution). The contribution percentages of KH2PO4 and Ca(NO3)2, pivotal macro-elements in Hoagland solution affecting duckweed protein content, were determined using Plackett-Burman factorial design as 33.06 % and 36.61 %, respectively. Additionally, optimization was conducted employing response surface methodology, incorporating pH alongside KH2PO4 and Ca(NO3)2. Under optimal conditions of 3.92 mM KH2PO4, 7.95 mM Ca(NO3)2, and 7.22 pH, the protein content of duckweed increased significantly, reaching 51.09 % from 39.81 %. The duckweed cultivated in modified Hoagland solution exhibited protein content of 41.74 %, while duckweed grown in commercial Hoagland solution displayed protein content of 33.01 %. This study showed protein content of duckweed could significantly increase according to the growth medium and showcasing its potential as a sustainable source of plant protein.

Fumaric acid production by Rhizopus species from acid hydrolysate of oil palm empty fruit bunches.

The hemicellulosic fraction of lignocellulosic biomass is a very important material, due to the significant concentration of pentoses present in its composition and that can be used sustainably in biotechnological processes such as the production of fumaric acid. Research efforts are currently being promoted for the proper disposal and valorization of empty fruit bunches (EFB) from oil palm. In this work, seventeen Rhizopus species were evaluated in a fermentation medium with EFB hydrolyzate, without detoxification, as a carbon source for fumaric acid production. Rhizopus circicans 1475 and Rhizopus 3271 achieved productions of 5.65 g.L-1 and 5.25 g.L-1 of fumaric acid at 30 °C, 120 rpm for 96 h, respectively. The percentage of consumed sugars, mainly pentoses, was 24.88% and 34.02% for R. circicans 1475 and R 3271, respectively. Soy peptone and ammonium sulfate were evaluated as nitrogen sources, where soy peptone stimulated the formation of biomass pellets while ammonium sulfate produced mycelia and clamps.

Comparative Analysis of Effect of Culture Conditions on Growth and C-Phycocyanin Production in Helical and Linear Spirulina.

Spirulina (Arthrospira and Spirulina spp.) has always been characterized by the helical trichomes, despite the existence of linear forms. A great debate is now open on the morphological flexibility of Spirulina, but it seems that both trichome morphology and C-phycocyanin (C-PC) concentrations are influenced by the culture conditions.This work compared the effect of some key growth factors (medium pH as well as its carbon, potassium, and salt contents) on the growth and C-PC concentration of helical and linear Spirulina strains. Further, two-phase strategies, including light and nitrogen variation, were applied to increase the in vivo C-PC accumulation into the trichomes. Results showed that high pH induced trichomes elongation and improved growth but decreased C-PC content (+ 65 and + 43% vs. -83 and -49%, for helical and linear strains, respectively). Variations in carbon and salt concentrations negatively impacted growth and C-PC content, even if the linear strain was more robust against these fluctuations. It was also interesting to see that potassium increasing improved growth and C-PC content for both strains.The variation of light wavelength during the enrichment phase (in the two-phase strategy) improved by 50% C-PC accumulation in trichomes, especially after blue lighting for 96 h. Similar result was obtained after 48 h of nitrogen reduction, while its removal from the medium caused trichomes disintegration. The current work highlights the robustness of linear Spirulina strain and presents an efficient and scalable way to increase C-PC in vivo without affecting growth.

Deletion of yghZ in Escherichia coli promotes growth in presence of furfural with xylose as carbon source.

Thermo-acidic pretreatment of lignocellulosic biomass is required to make it amenable to microbial metabolism and results in generation of furfural due to breakdown of pentose sugars. Furfural is toxic to microbial metabolism and results in reduced microbial productivity and increased production costs. This study asks if deletion of yghZ gene which encodes a NADPH-dependent aldehyde reductase enzyme results in improved furfural tolerance in Escherichia coli host. The ∆yghZ strain-SSK201-was tested for tolerance to furfural in presence of 5% xylose as a carbon source in AM1 minimal medium. At 96 h and in presence of 1.0 g/L furfural, the culture harboring strain SSK201 displayed 4.5-fold higher biomass, 2-fold lower furfural concentration and 15.75-fold higher specific growth rate (µ) as compared to the parent strain SSK42. The furfural tolerance advantage of SSK201 was retained when the carbon source was switched to glucose in AM1 medium and was lost in rich LB medium. The findings have potential to be scaled up to a hydrolysate culture medium, which contains furan inhibitors and lack nutritionally rich components, under bioreactor cultivation and observe growth advantage of the ∆yghZ host. It harbors potential to generate robust industrial strains which can convert lignocellulosic carbon into metabolites of interest in a cost-efficient manner.

Comprehensive assessment of goat adipose tissue-derived mesenchymal stem cells cultured in different media.

Mesenchymal stem cells (MSCs) have demonstrated potential in treating livestock diseases that are unresponsive to conventional therapies. MSCs derived from goats, a valuable model for studying orthopaedic disorders in humans, offer insights into bone formation and regeneration. Adipose tissue-derived MSCs (ADSCs) are easily accessible and have a high capacity for expansion. Although the choice of culture media significantly influences the biological properties of MSCs, the optimal media for goat ADSCs (gADSCs) remains unclear. This study aimed to assess the effects of four commonly used culture media on gADSCs' culture characteristics, stem cell-specific immunophenotype, and differentiation. Results showed that MEM, DMEM/F12, and DMEM-LG were superior in maintaining cell morphology and culture parameters of gADSCs, such as cell adherence, metabolic activity, colony-forming potential, and population doubling. Conversely, DMEM-HG exhibited poor performance across all evaluated parameters. The gADSCs cultured in DMEM/F12 showed enhanced early proliferation and lower apoptosis. The cell surface marker distribution exhibited superior characteristics in gADSCs cultured in MEM and DMEM/F12. In contrast, the distribution was inferior in gADSCs cultured in DMEM-LG. DMEM/F12 and DMEM-LG culture media demonstrated a significantly higher potential for chondrogenic differentiation and DMEM-LG for osteogenic differentiation. In conclusion, DMEM/F12 is a suitable culture medium for propagating gADSCs as it effectively maintains cell morphology, growth parameters, proliferation and lower apoptosis while exhibiting desirable expression patterns of MSC-specific markers. These findings contribute to optimising culture conditions for gADSCs, enhancing their potential applications in disease treatment and regenerative medicine.

An Optimized Human Erythroblast Differentiation System Reveals Cholesterol-Dependency of Robust Production of Cultured Red Blood Cells Ex Vivo.

The generation of cultured red blood cells (cRBCs) ex vivo represents a potentially unlimited source for RBC transfusion and other cell therapies. Human cRBCs can be generated from the terminal differentiation of proliferating erythroblasts derived from hematopoietic stem/progenitor cells or erythroid precursors in peripheral blood mononuclear cells. Efficient differentiation and maturation into cRBCs highly depend on replenishing human plasma, which exhibits variable potency across donors or batches and complicates the consistent cRBC production required for clinical translation. Hence, the role of human plasma in erythroblast terminal maturation is investigated and uncovered that 1) a newly developed cell culture basal medium mimicking the metabolic profile of human plasma enhances cell growth and increases cRBC yield upon erythroblast terminal differentiation and 2) LDL-carried cholesterol, as a substitute for human plasma, is sufficient to support erythroid survival and terminal differentiation ex vivo. Consequently, a chemically-defined optimized medium (COM) is developed, enabling robust generation of cRBCs from erythroblasts of multiple origins, with improved enucleation efficiency and higher reticulocyte yield, without the need for supplementing human plasma or serum. In addition, the results reveal the crucial role of lipid metabolism during human terminal erythropoiesis.

Genome-scale metabolic modeling of Thraustochytrium sp. RT2316-16: Effects of nutrients on metabolism.

Marine thraustochytrids produce metabolically important lipids such as the long-chain omega-3 polyunsaturated fatty acids, carotenoids, and sterols. The growth and lipid production in thraustochytrids depends on the composition of the culture medium that often contains yeast extract as a source of amino acids. This work discusses the effects of individual amino acids provided in the culture medium as the only source of nitrogen, on the production of biomass and lipids by the thraustochytrid Thraustochytrium sp. RT2316-16. A reconstructed metabolic network based on the annotated genome of RT2316-16 in combination with flux balance analysis was used to explain the observed growth and consumption of the nutrients. The culture kinetic parameters estimated from the experimental data were used to constrain the flux via the nutrient consumption rates and the specific growth rate of the triacylglycerol-free biomass in the genome-scale metabolic model (GEM) to predict the specific rate of ATP production for cell maintenance. A relationship was identified between the specific rate of ATP production for maintenance and the specific rate of glucose consumption. The GEM and the derived relationship for the production of ATP for maintenance were used in linear optimization problems, to successfully predict the specific growth rate of RT2316-16 in different experimental conditions.

Catechins prevent monoclonal antibody fragmentation during production via fed-batch culture of Chinese hamster ovary cells.

Chinese hamster ovary (CHO) cells are widely used for the industrial production of therapeutic monoclonal antibodies (mAbs). To meet the increasing market demands, high productivity, and quality are required in cell culture. One of the critical attributes of mAbs, from a safety perspective, is mAb fragmentation. However, methods for preventing mAbs fragmentation in CHO cell culture are limited. In this study, we observed that the antibody fragment content increased with increasing titers in fed-batch cultures for all three cell lines expressing recombinant antibodies. Adding copper sulfate to the culture medium further increased the fragment content, suggesting the involvement of reactive oxygen species (ROS) in the fragmentation process. Though antioxidants may be helpful to scavenge ROS, several antioxidants are reported to decrease the productivity of CHO cells. Among the antioxidants examined, we observed that the addition of catechin or (-)-epigallocatechin gallate to the culture medium prevented fragmentation content by about 20% and increased viable cell density and titer by 30% and 10%, respectively. Thus, the addition of catechins or compounds of equivalent function would be beneficial for manufacturing therapeutic mAbs with a balance between high titers and good quality.

Pseudomonas aeruginosa transcriptome analysis of metal restriction in ex vivo cystic fibrosis sputum.

Chronic Pseudomonas aeruginosa lung infections are a feature of cystic fibrosis (CF) that many patients experience even with the advent of highly effective modulator therapies. Identifying factors that impact P. aeruginosa in the CF lung could yield novel strategies to eradicate infection or otherwise improve outcomes. To complement published P. aeruginosa studies using laboratory models or RNA isolated from sputum, we analyzed transcripts of strain PAO1 after incubation in sputum from different CF donors prior to RNA extraction. We compared PAO1 gene expression in this "spike-in" sputum model to that for P. aeruginosa grown in synthetic cystic fibrosis sputum medium to determine key genes, which are among the most differentially expressed or most highly expressed. Using the key genes, gene sets with correlated expression were determined using the gene expression analysis tool eADAGE. Gene sets were used to analyze the activity of specific pathways in P. aeruginosa grown in sputum from different individuals. Gene sets that we found to be more active in sputum showed similar activation in published data that included P. aeruginosa RNA isolated from sputum relative to corresponding in vitro reference cultures. In the ex vivo samples, P. aeruginosa had increased levels of genes related to zinc and iron acquisition which were suppressed by metal amendment of sputum. We also found a significant correlation between expression of the H1-type VI secretion system and CFTR corrector use by the sputum donor. An ex vivo sputum model or synthetic sputum medium formulation that imposes metal restriction may enhance future CF-related studies.IMPORTANCEIdentifying the gene expression programs used by Pseudomonas aeruginosa to colonize the lungs of people with cystic fibrosis (CF) will illuminate new therapeutic strategies. To capture these transcriptional programs, we cultured the common P. aeruginosa laboratory strain PAO1 in expectorated sputum from CF patient donors. Through bioinformatic analysis, we defined sets of genes that are more transcriptionally active in real CF sputum compared to a synthetic cystic fibrosis sputum medium. Many of the most differentially active gene sets contained genes related to metal acquisition, suggesting that these gene sets play an active role in scavenging for metals in the CF lung environment which may be inadequately represented in some models. Future studies of P. aeruginosa transcript abundance in CF may benefit from the use of an expectorated sputum model or media supplemented with factors that induce metal restriction.

Functional and structural responses of a halophilic consortium to oily sludge during biodegradation.

Biotreatment of oily sludge and the involved microbial communities, particularly in saline environments, have been rarely investigated. We enriched a halophilic bacterial consortium (OS-100) from petroleum refining oily sludge, which degraded almost 86% of the aliphatic hydrocarbon (C10-C30) fraction of the oily sludge within 7 days in the presence of 100 g/L NaCl. Two halophilic hydrocarbon-degrading bacteria related to the genera Chromohalobacter and Halomonas were isolated from the OS-100 consortium. Hydrocarbon degradation by the OS-100 consortium was relatively higher compared to the isolated bacteria, indicating potential synergistic interactions among the OS-100 community members. Exclusion of FeCl2, MgCl2, CaCl2, trace elements, and vitamins from the culture medium did not significantly affect the hydrocarbon degradation efficiency of the OS-100 consortium. To the contrary, hydrocarbon biodegradation dropped from 94.1 to 54.4% and 5% when the OS-100 consortium was deprived from phosphate and nitrogen sources in the culture medium, respectively. Quantitative PCR revealed that alkB gene expression increased up to the 3rd day of incubation with 11.277-fold, consistent with the observed increments in hydrocarbon degradation. Illumina-MiSeq sequencing of 16 S rRNA gene fragments revealed that the OS-100 consortium was mainly composed of the genera Halomonas, Idiomarina, Alcanivorax and Chromohalobacter. This community structure changed depending on the culturing conditions. However, remarkable changes in the community structure were not always associated with remarkable shifts in the hydrocarbonoclastic activity and vice versa. The results show that probably synergistic interactions between community members and different subpopulations of the OS-100 consortium contributed to salinity tolerance and hydrocarbon degradation.

Transcriptomic profiling of adding cobalt chloride to improve dendrobine-type total alkaloid production.

Trichoderma longibrachiatum UN32 is known for its efficient production of dendrobine-type total alkaloids (DTTAs). This study aimed to determine the optimal medium composition for the UN32 strain using response surface methodology. Key factors, including glucose, beef extract, and CoCl2, were selected through the Plackett-Burman design. Subsequently, a factorial optimization approach was employed using the steepest ascent design, and 17 trial sets were completed via the Box-Behnken design. The optimal medium composition was found to consist of 29.4 g/L of glucose, 17.3 g/L of beef extract, and 0.28 mmol/L of CoCl2. This optimized medium resulted in an impressive 80.8% increase in mycelial dry weight (reaching 12.303 g/L) and a substantial 76.4% boost in DTTA production (reaching 541.63 ± 46.95 µg). Furthermore, the fermentation process was scaled up to a 5-L bioreactor, leading to a DTTA production approximately 1.95 times than the control. Transcriptome analysis of strain UN32 in response to CoCl2 supplementation revealed significant changes in the expression of critical genes associated with the TCA cycle and L-valine, L-leucine, and L-isoleucine biosynthesis changed. These alterations resulted in a heightened influx of acetyl-CoA into DTTA production. Additionally, the expression of genes related to antioxidant enzymes was modified to maintain homeostasis of reactive oxygen species (ROS). A potential mechanism for the accumulation of DTTAs based on ROS as a signal transduction was proposed. These findings provide valuable insights into the regulatory mechanisms of DTTA biosynthesis, potentially offering a method to enhance the production of secondary metabolites in the UN32 strain. KEY POINTS: • After the RSM optimization, there is a substantial increase of 80.8% in biomass production and a significant 76.4% rise in DTTA production. • Transcriptome analysis revealed that the inclusion of CoCl2 supplements resulted in an enhanced influx of acetyl-CoA. • Proposed a mechanism for the accumulation of DTTAs for the role of ROS as a signal transduction pathway.

Poly-γ-glutamic acid overproduction of Bacillus licheniformis ATCC 9945a by developing a novel optimum culture medium and glutamate pulse feeding using different experimental design approaches.

The commercial production of multifunctional, biocompatible, and biodegradable biopolymers such as poly-γ-glutamic acid via microbial fermentation requires the development of simple and cheap methods for mass production. This study optimized the poly-γ-glutamic acid production of Bacillus licheniformis ATCC 9945a in several steps. At first, the most critical components of the culture medium, including l-glutamic acid, citric acid, and glycerol, were selected by screening nine factors through the Plackett-Burman experimental design and then were optimized using the response surface method and the central composite design algorithm. Under optimal conditions, the production of poly-γ-glutamic acid increased by more than 4.2 times from 11.2 to 47.2 g/L. This is one of the highest production rates of this strain in submerged batch fermentation reported so far using the optimized medium compared to the conventional base medium. A novel and efficient sudden pulse feeding strategy (achieved by a novel one-factorial statistical technique) of l-glutamic acid to the optimized medium increased biopolymer production from 47.2 to 66.1 g/L, the highest value reported in published literature with this strain. This simple, reproducible, and cheap fermentation process can considerably enhance the commercial applications of the poly-γ-glutamic acid synthesized by B. licheniformis ATCC 9945a.

Dynamic flux balance analysis of high cell density fed-batch culture of Escherichia coli BL21 (DE3) with mass spectrometry-based spent media analysis.

Dynamic flux balance analysis (FBA) allows estimation of intracellular reaction rates using organism-specific genome-scale metabolic models (GSMM) and by assuming instantaneous pseudo-steady states for processes that are inherently dynamic. This technique is well-suited for industrial bioprocesses employing complex media characterized by a hierarchy of substrate uptake and product secretion. However, knowledge of exchange rates of many components of the media would be required to obtain meaningful results. Here, we performed spent media analysis using mass spectrometry coupled with liquid and gas chromatography for a fed-batch, high-cell density cultivation of Escherichia coli BL21(DE3) expressing a recombinant protein. Time course measurements thus obtained for 246 metabolites were converted to instantaneous exchange rates. These were then used as constraints for dynamic FBA using a previously reported GSMM, thus providing insights into how the flux map evolves through the process. Changes in tri-carboxylic acid cycle fluxes correlated with the increased demand for energy during recombinant protein production. The results show how amino acids act as hubs for the synthesis of other cellular metabolites. Our results provide a deeper understanding of an industrial bioprocess and will have implications in further optimizing the process.

Inhibition of Polyphosphate Degradation in Synechocystis sp. PCC6803 through Inactivation of the phoU Gene.

Phosphorus is an essential but non-renewable nutrient resource critical for agriculture. Luxury phosphorus uptake allows microalgae to synthesize polyphosphate and accumulate phosphorus, but, depending on the strain of algae, polyphosphate may be degraded within 4 hours of accumulation. We studied the recovery of phosphorus from wastewater through luxury uptake by an engineered strain of Synechocystis sp. with inhibited polyphosphate degradation and the effect of this engineered Synechocystis biomass on lettuce growth. First, a strain (ΔphoU) lacking the phoU gene, which encodes a negative regulator of environmental phosphate concentrations, was generated to inhibit polyphosphate degradation in cells. Polyphosphate concentrations in the phoU knock-out strain were maintained for 24 h and then decreased slowly. In contrast, polyphosphate concentrations in the wild-type strain increased up to 4 h and then decreased rapidly. In addition, polyphosphate concentration in the phoU knockout strain cultured in semi-permeable membrane bioreactors with artificial wastewater medium was 2.5 times higher than that in the wild type and decreased to only 16% after 48 h. The biomass of lettuce treated with the phoU knockout strain (0.157 mg P/m2) was 38% higher than that of the lettuce treated with the control group. These results indicate that treating lettuce with this microalgal biomass can be beneficial to crop growth. These results suggest that the use of polyphosphate-accumulating microalgae as biofertilizers may alleviate the effects of a diminishing phosphorous supply. These findings can be used as a basis for additional genetic engineering to increase intracellular polyphosphate levels.

Initial culture media pH influences the antibacterial activity and metabolic footprint of Lactobacillus acidophilus BIOTECH 1900.

This study investigated the impact of initial culture media pH on the antibacterial properties and metabolic profile of cell-free supernatants (CFSs) from Lactobacillus acidophilus BIOTECH 1900 (LAB1900). The CFSs harvested from LAB1900 grown in de Man, Rogosa, Sharpe broth with initial pH of 5.5 (CFS5.5) and 6.6 (CFS6.6) were tested. The two CFSs elicited varying degrees of activity against three gram-negative bacteria. In the agar-well diffusion against Pseudomonas aeruginosa, CFS5.5 and CFS6.6 recorded 14.36 ± 1.34 and 13.06 ± 1.29 mm inhibition, respectively. Interestingly, against Klebsiella pneumoniae, CFS5.5 showed 14.36 ± 1.56 mm inhibition which was significantly higher than the 12.22 ± 1.31 mm inhibition of CFS6.6 (p = 0.0464). While against Acinetobacter baumannii, significantly higher inhibition of 10.66 ± 0.51 mm was observed in CFS6.6 compared to the 7.58 ± 1.93 mm inhibition of CFS5.5 (p = 0.0087). Nonetheless, both CFSs were bactericidal, with a minimum inhibitory and bactericidal concentration range of 3.90625-7.8125 mg/mL. The varied antibacterial activities may be attributed to the metabolite compositions of CFSs. A total of 152 metabolites driving the separation between CFSs were noted, with the majority upregulated in CFS5.5. Furthermore, 15 were putatively identified belonging to acylcarnities, vitamins, gibberellins, glycerophospholipids, and peptides. In summary, initial culture media pH affects the production of microbial metabolites with antibacterial properties.

Challenges in developing cell culture media using machine learning.

Microbial and mammalian cells are widely used in the food, pharmaceutical, and medical industries. Developing or optimizing culture media is essential to improve cell culture performance as a critical technology in cell culture engineering. Methodologies for media optimization have been developed to a great extent, such as the approaches of one-factor-at-a-time (OFAT) and response surface methodology (RSM). The present review introduces the emerging machine learning (ML) technology in cell culture engineering by combining high-throughput experimental technologies to develop highly efficient and effective culture media. The commonly used ML algorithms and the successful applications of employing ML in medium optimization are summarized. This review highlights the benefits of ML-assisted medium development and guides the selection of the media optimization method appropriate for various cell culture purposes.

Media matters: culture medium-dependent hypervariable phenotype of mesenchymal stromal cells.

BACKGROUND: Despite a long history of investigation and sustained efforts in clinical testing, the number of market authorisations for mesenchymal stromal cell (MSC) therapies remains limited, with none approved by the United States Food and Drug Administration. Several barriers are impeding the clinical progression of MSC therapies, to the forefront of these is a lack of standardised manufacturing protocols which is further compounded by an absence of biologically meaningful characterisation and release assays. A look at clinical trial registries demonstrates the diversity of MSC expansion protocols with variabilities in cell source, isolation method and expansion medium, among other culture variables, making it extraordinarily difficult to compare study outcomes. Current identification and characterisation standards are insufficient; they are not specific to MSCs and do not indicate cell function or therapeutic action. METHODS: This work analysed the influence of five widely used culture media formulations on the colony-forming potential, proliferation kinetics, trilineage differentiation potential and immunomodulatory potential of human bone marrow-derived MSCs (BM-MSCs). The surface marker expression profiles were also characterised using a high-content flow cytometry screening panel of 243 markers. RESULTS: Significant differences in the biological attributes of BM-MSCs including clonogenicity, proliferation, differentiation propensity and immunomodulatory capacity were revealed in response to the composition of the culture medium. Despite their biological differences, all cell preparations uniformly and strongly expressed the standard positive markers proposed for BM-MSCs: CD73, CD90 and CD105. Immunophenotypic profiling revealed that the culture medium also had a significant influence on the surface proteome, with one-third of tested markers exhibiting variable expression profiles. Principal component analysis demonstrated that BM-MSCs isolated and expanded in a proprietary xeno- and serum-free medium displayed the most consistent cell phenotypes with little variability between donors compared to platelet lysate and foetal bovine serum-containing media. CONCLUSIONS: These data suggest that media composition has a highly significant impact on the biological attributes of MSCs, but standard surface marker tests conceal these differences. The results indicate a need for (1) standardised approaches to manufacturing, with an essential focus on defined media and (2) new biologically relevant tests for MSC characterisation and product release.