Final Cell Density Research Articles

Large-Scale Expansion of Human Liver Stem Cells Using Two Different Bioreactor Systems.

The assessment of human liver stem cells (HLSCs) as cell therapeutics requires scalable, controlled expansion processes. We first focused on defining appropriate process parameters for HLSC expansion such as seeding density, use of antibiotics, optimal cell age and critical metabolite concentrations in conventional 2D culture systems. For scale-up, we transferred HLSC expansion to multi-plate and stirred-tank bioreactor systems to determine their limitations. A seeding density of 4000 cells cm-2 was needed for efficient expansion. Although growth was not significantly affected by antibiotics, the concentrations of lactate and ammonia were important. A maximum expansion capacity of at least 20 cumulative population doublings (cPDs) was observed, confirming HLSC growth, identity and functionality. For the expansion of HLSCs in the multi-plate bioreactor system Xpansion (XPN), the oxygen supply strategy was optimized due to a low kLa of 0.076 h-1. The XPN bioreactor yielded a final mean cell density of 94 ± 8 × 103 cells cm-2, more than double that of the standard process in T-flasks. However, in the larger XPN50 device, HLSC density reached only 28 ± 0.9 × 103 cells cm-2, while the glucose consumption rate increased 8-fold. In a fully-controlled 2 L stirred-tank bioreactor (STR), HLSCs expanded at a comparable rate to the T-flask and XPN50 processes in a homogeneous microenvironment using advanced process analytical technology. Ultimately, the scale-up of HLSCs was successful using two different bioreactor systems, resulting in sufficient numbers of viable, functional and undifferentiated HLSCs for therapeutic applications.

Morphology, Phylogeny and Discovery of the Antioxidant Potential of a Marine Dinoflagellate Karlodinium ballantinum (Kareniaceae, Dinophyceae) Isolated from Subic Bay, Zambales, Central Luzon, Philippines

Morphological characteristics, phylogenetic analysis, and antioxidant activity of a marine unarmored dinoflagellate Karlodinium ballantinum were examined using a culture established from Subic Bay, Zambales in November 2022. Detailed examinations were based on light microscopy and molecular phylogeny inferred from internal transcribed spacer (ITS) and LSU rDNA sequences. Cells of K. ballantinum were small and ellipsoid, possessing a straight ASC, large central nucleus, accumulation bodies, and unequally distributed chloroplasts with internal pyrenoids. Cell size ranged from 7.2–13.7 μm in length and 5.4–9.0 μm in width. Phylogenetic analyses suggested that K. ballantinum has a close affinity to K. gentienii and K. zhouanum. Upscaling the culture into 20 L, the final cell density of the culture reached approximately 177.5×106 cells/mL, which produced 0.1 g of algal biomass. The antioxidant activity of K. ballantinum, as screened using the DPPH assay, showed a 23.39% antioxidative property. This result displayed the potential of K. ballantinum to be beneficial microalgae despite being known as a causative agent of HABs. The present study is the first record of K. ballantinum in Subic Bay, Zambales, and serves as the first report of the antioxidant activity of a species under the genus Karlodinium.

Effects of methane to oxygen ratio on cell growth and polyhydroxybutyrate synthesis in high cell density cultivation of Methylocystis sp. MJC1.

Polyhydroxybutyrate (PHB) production through CH4 conversion by methanotrophs offers a solution for greenhouse gas emissions and plastic waste concerns. In this study, we aimed to achieve high cell density cultivation of Methylocystis sp. MJC1 for efficient PHB production. Cultivating MJC1 using CH4 and air (3:7, v/v) yielded a final cell density of 52.9 g/L with a 53.7% (28.4 g/L) PHB content after 210 h, showcasing PHB mass production potential. However, long-term cultivation led to a low volumetric productivity of 0.200g/L/h. To address this, we conducted cultivation at various O2/CH4 ratios using O2 instead of air, which significantly improved the PHB productivity. Under high O2 conditions (O2/CH4 ratio of 1.5), biomass productivity increased 1.51-fold compared to that under low O2 conditions in the same time frame; however, PHB accumulation was delayed. Using an equal ratio of CH4 and O2 induced active cell growth and selective PHB production, achieving the highest PHB productivity (0.365 g/L/h) with a final cell density of 55.9 g/L and PHB content of 61.7% (34.5 g/L) in 162 h. This study highlighted the significance of the O2/CH4 ratio in CH4 conversion and PHB production by M. sp. MJC1.

COSMIC-dFBA: A novel multi-scale hybrid framework for bioprocess modeling

Metabolism governs cell performance in biomanufacturing, as it fuels growth and productivity. However, even in well-controlled culture systems, metabolism is dynamic, with shifting objectives and resources, thus limiting the predictive capability of mechanistic models for process design and optimization. Here, we present Cellular Objectives and State Modulation In bioreaCtors (COSMIC)-dFBA, a hybrid multi-scale modeling paradigm that accurately predicts cell density, antibody titer, and bioreactor metabolite concentration profiles. Using machine-learning, COSMIC-dFBA decomposes the instantaneous metabolite uptake and secretion rates in a bioreactor into weighted contributions from each cell state (growth or antibody-producing state) and integrates these with a genome-scale metabolic model. A major strength of COSMIC-dFBA is that it can be parameterized with only metabolite concentrations from spent media, although constraining the metabolic model with other omics data can further improve its capabilities. Using COSMIC-dFBA, we can predict the final cell density and antibody titer to within 10% of the measured data, and compared to a standard dFBA model, we found the framework showed a 90% and 72% improvement in cell density and antibody titer prediction, respectively. Thus, we demonstrate our hybrid modeling framework effectively captures cellular metabolism and expands the applicability of dFBA to model the dynamic conditions in a bioreactor.

Growth prolongation of human induced pluripotent stem cell aggregate in three-dimensional suspension culture system by addition of botulinum hemagglutinin

Human induced pluripotent stem cells (hiPSCs) can be used in regenerative therapy as an irresistible cell source, and so the development of scalable production of hiPSCs for three-dimensional (3D) suspension culture is required. In this study, we established a simple culture strategy for improving hiPSC aggregate growth using botulinum hemagglutinin (HA), which disrupts cell-cell adhesion mediated by E-cadherin. When HA was added to the suspension culture of hiPSC aggregates, E-cadherin-mediated cell-cell adhesion was temporarily disrupted within 24h, but then recovered. Phosphorylated myosin light chain, a contractile force marker, was also recovered at the periphery of hiPSC aggregates. The cell aggregates were suppressed the formation of collagen type I shell-like structures at the periphery by HA and collagen type I was homogenously distributed within the cell aggregates. In addition, these cell aggregates retained the proliferation marker Ki-67 throughout the cell aggregates. The apparent specific growth rate with HA addition was maintained continuously throughout the culture, and the final cell density was 1.7-fold higher than that in the control culture. These cells retained high expression levels of pluripotency markers. These observations indicated that relaxation of cell-cell adhesions by HA addition induced rearrangement of the mechanical tensions generated by actomyosin in hiPSC aggregates and suppression of collagen type I shell-like structure formation. These results suggest that this simple and readily culture strategy is a potentially useful tool for improving the scalable production of hiPSCs for 3D suspension cultures.

Computer controlled expansion of equine cord blood mesenchymal stromal cells on microcarriers in 3L vertical-wheel® bioreactors.

Mesenchymal stromal cells (MSCs) are an ideal cell source for allogenic cell therapy due to their immunomodulatory and differentiation properties. Equine MSCs (eMSCs) have been found to be a promising treatment for equine joint injuries including meniscal injuries, cartilage degradation, and osteoarthritis. Although the use of eMSCs has shown efficacy in preliminary studies, challenges associated with biomanufacturing remain. To achieve the required cell numbers for clinical application, bioreactor-based processes are required. Initial studies have shown that eMSCs can be cultivated in microcarrier-based, stirred suspension bioreactor culture at the laboratory 0.1L scale using a Vertical-Wheel® (VW) bioreactor. However, investigations regarding scale up of these processes to the required biomanufacturing scales are required. This study investigated the scale-up of a equine cord blood MSC (eCB-MSC) bioprocess in VW bioreactors at three scales. This included scale-up from the 0.1-0.5L bioreactor, scale-up from static culture to the 3L computer-controlled bioreactor, and scale-up into the 3L computer-controlled bioreactor using a mock clinical trial process. Results from the various scale-up experiments demonstrated similar cell expansion at the various tested scales. The 3L computer-controlled system resulted in a final cell densities of 1.5 × 105 cells/cm2 on average, achieving 1.5 × 109 harvested cells. Biological testing of the cells showed that cell phenotype and functionality were maintained after scale-up. These findings demonstrate the scalability of an eCB-MSC bioprocess using microcarriers in VW bioreactors to achieve clinically relevant cell numbers, a critical step to translate MSC treatments from research to clinical applications. This study also represents the first known published study expanding any cell type in the 3L VW bioreactor.

Towards universal synthetic heterotrophy using a metabolic coordinator

Engineering the utilization of non-native substrates, or synthetic heterotrophy, in proven industrial microbes such as Saccharomyces cerevisiae represents an opportunity to valorize plentiful and renewable sources of carbon and energy as inputs to bioprocesses. We previously demonstrated that activation of the galactose (GAL) regulon, a regulatory structure used by this yeast to coordinate substrate utilization with biomass formation during growth on galactose, during growth on the non-native substrate xylose results in a vastly altered gene expression profile and faster growth compared with constitutive overexpression of the same heterologous catabolic pathway. However, this effort involved the creation of a xylose-inducible variant of Gal3p (Gal3pSyn4.1), the sensor protein of the GAL regulon, preventing this semi-synthetic regulon approach from being easily adapted to additional non-native substrates. Here, we report the construction of a variant Gal3pMC (metabolic coordinator) that exhibits robust GAL regulon activation in the presence of structurally diverse substrates and recapitulates the dynamics of the native system. Multiple molecular modeling studies suggest that Gal3pMC occupies conformational states corresponding to galactose-bound Gal3p in an inducer-independent manner. Using Gal3pMC to test a regulon approach to the assimilation of the non-native lignocellulosic sugars xylose, arabinose, and cellobiose yields higher growth rates and final cell densities when compared with a constitutive overexpression of the same set of catabolic genes. The subsequent demonstration of rapid and complete co-utilization of all three non-native substrates suggests that Gal3pMC-mediated dynamic global gene expression changes by GAL regulon activation may be universally beneficial for engineering synthetic heterotrophy.

Optimization of a hybrid bacterial/Arabidopsis thaliana fatty acid synthase system II in Saccharomyces cerevisiae

Fatty acids are produced by eukaryotes like baker's yeast Saccharomyces cerevisiae mainly using a large multifunctional type I fatty acid synthase (FASI) where seven catalytic steps and a carrier domain are shared between one or two protein subunits. While this system may offer efficiency in catalysis, only a narrow range of fatty acids are produced. Prokaryotes, chloroplasts and mitochondria rely instead on a FAS type II (FASII) where each catalytic step is carried out by a monofunctional enzyme encoded by a separate gene. FASII is more flexible and capable of producing a wider range of fatty acid structures, such as the direct production of unsaturated fatty acids. An efficient FASII in the preferred industrial organism S. cerevisiae could provide a platform for developing sustainable production of specialized fatty acids. We functionally replaced either yeast FASI genes (FAS1 or FAS2) with a FASII consisting of nine genes from Escherichia coli (acpP, acpS and fab -A, -B, -D, -F, -G, -H, -Z) as well as three from Arabidopsis thaliana (MOD1, FATA1 and FATB). The genes were expressed from an autonomously replicating multicopy vector assembled using the Yeast Pathway Kit for in-vivo assembly in yeast. Two rounds of adaptation led to a strain with a maximum growth rate (μmax) of 0.19 h−1 without exogenous fatty acids, twice the growth rate previously reported for a comparable strain. Additional copies of the MOD1 or fabH genes resulted in cultures with higher final cell densities and three times higher lipid content compared to the control.

Pairing Colicins B and E5 with Bdellovibrio bacteriovorus To Eradicate Carbapenem- and Colistin-Resistant Strains of Escherichia coli.

While diverse antibacterials are available in nature, each possesses their own strengths and limitations. One such antibacterial is colicins, proteinaceous toxins that are produced by strains of E. coli to subvert the growth or viability of other E. coli strains. Similarly, predatory bacteria, of which Bdellovibrio bacteriovorus is well-known, are microbes that actively predate on and consume other Gram-negative bacterial strains. While they are all quite active as antibacterials, they also present some limitations: rapid resistance development to colicins while predation does not completely kill their prey. Within this study, therefore, we evaluated the impact of two different colicins (colicin B [ColB] and colicin E5 [ColE5]) and B. bacteriovorus HD100 either individually or together against four clinical isolates of E. coli that are resistant to either colistin or carbapenem. While the ColB and ColE5 were quickly active when used alone, causing a significant loss in viability (>3-log) in susceptible populations after only 3 h, the pathogens always grew afterwards and had final cell densities that were similar with their respective controls. Predation with B. bacteriovorus HD100, in contrast, was most pronounced after 24 h (>3-log reduction in each pathogen viability but never complete). When combined, better killing efficiencies were observed with several of the pathogens, with complete eradication realized for two (<100 viable pathogens per mL). Given the diversity of colicins in nature and the broad-spectrum activities of B. bacteriovorus strains, the results presented here suggest there is a massive potential to control pathogens when they are used together. IMPORTANCE The coupled impact of drug resistance with reduced antibiotic development has placed humankind at a postantibiotic crossroads where antibiotic alternatives are desperately needed. Consequently, we discuss here the combined effectiveness of two vastly different classes of antibacterials, namely, colicins and a predatory bacterium (i.e.,Bdellovibrio bacteriovorus HD100), against two priority pathogenic groups, colistin- and carbapenem-resistant strains of E. coli. While each is effective in its own manner, these antibacterials also display limitations, i.e., the rapid appearance of mutations that confer resistance to the colicins while predatory bacteria do not completely kill their prey. Here, we show these limitations can be overcome using combined treatments of these antibacterials, with two pathogenic E. coli populations completely eradicated within 24 h. Given the diversity of colicins and the broad-spectrum activities of B. bacteriovorus strains, the results presented here suggests there is a massive potential to control pathogens when they are used together.

High-Cell-Density Yeast Oil Production with Diluted Substrates Imitating Microalgae Hydrolysate Using a Membrane Bioreactor

Microbial oil production from industrial waste streams and biogenic resources, such as biomass hydrolysates, is emerging as a sustainable alternative to use of fossil and vegetable oils. However, the carbon sources of these substrates are typically diluted, leading to low product concentrations and, therefore, high fermentation and downstream processing costs. In this study, high-cell-density yeast oil production with a defined medium, which imitated the sugar composition of a diluted substrate, a typical microalgal biomass hydrolysate, is carried out on a 50 L scale using a membrane bioreactor (MBR) consisting of a microfiltration unit suited for industrial application. The process was run on a semi-continuous mode to reduce operational costs. Oleaginous yeast Cutaneotrichosporon oleaginosus was used as a biocatalyst and lipid production was induced by phosphate deficiency in the medium with a C/P ratio of 3515 g g−1. In this way, high cellular lipid contents of up to 76.5% (w/w) of dry cell mass, an average lipid yield of 32% (w/w), and a lipid space–time yield (STY) of up to 8.88 g L−1 d−1 were achieved with final high cell densities of up to 116 g L−1 dry biomass. Furthermore, use of a defined medium and elemental analysis of the yeast cells and yeast oil enabled drawing an accurate carbon mass balance of the production system. Carbon conversion efficiencies—fraction of total carbon supplied in the form of sugars converted into lipids at the end of the process—of up to 61.5% were achieved from diluted substrates using the MBR with total cell retention. Considering these results, it is concluded that utilization of an MBR on a semi-continuous mode would be very reasonable for yeast oil production, enabling high productivities with diluted sugar substrates.

Biological role of the major AP (abasic site) endonuclease of an archaeon from geothermal environments.

Archaea and bacteria in geothermal environments are predicted to suffer DNA depurination in vivo at high rates, which raises questions regarding the biological roles of their abasic-site-repair enzymes. Gene deletion and enzymatic assay demonstrated that the saci_0015 gene of Sulfolobus acidocaldarius encodes an AP endonuclease (Apn) accounting for as much as 95% of the assayable activity in cell extracts and is not essential for viability. To identify genetic functions of this enzyme, deletion (ΔApn) strains were examined with respect to growth, spontaneous mutation, transformation by ssDNA containing an abasic site, and conjugation. Relative to its isogenic control, the ΔApn strain did not exhibit any change in growth rate or final cell density, rate or spectrum of spontaneous mutation, transformation by DNA containing an abasic site, or efficiency of DNA transfer and recombination. The apparent lack of genetic impact of removing the major AP endonuclease was unexpected and indicated that abasic sites are rarely bypassed directly by DNA polymerases in S. acidocaldarius. AP endonuclease deficiency had no obvious effect on survival of S. acidocaldarius under several test conditions, but it accelerated the death of cells at 4º C under illumination. Our results suggest that the normal level of AP endonuclease in S. acidocaldarius is well above the minimum required for growth and cell division but not for recovery from prolonged exposure to certain low-temperature conditions. This situation illustrates a biological challenge that has not been emphasized in experimental studies of extremophiles, i.e., the problem of long-term survival under "non-extreme" conditions.

The Production of γ-Aminobutyric Acid from Free and Immobilized Cells of Levilactobacillus brevis Cultivated in Anaerobic and Aerobic Conditions.

γ-aminobutyric acid (GABA) has several beneficial effects on human health. GABA may be produced via chemical synthesis or through microbial metabolism, and Levilactobacillus brevis is recognized as a GABA-producing species. In this study, 11 Lvb. brevis strains were screened for GABA production, and the best producers were selected to verify the effect of aerobic (AE) and respiratory (RS) cultivations on growth parameters, biomass, and GABA accumulation. Lvb. brevis LB12 was then used to evaluate the combined effect of the incubation atmosphere (anaerobiosis vs. aerobiosis), cell protection (free vs. immobilized cells), and cell recycling (fresh vs. starved cells) on GABA production. Glutamate (GLU) consumption and GABA accumulation were detected by Thin-layer Chromatography (TLC) and RP-HPLC analyses. The ability to produce GABA was widespread among the strains. AE and RS growth improved biomass production, but oxygen availability impaired GLU to GABA conversion, and the anaerobically growing cells had the highest GABA productivity. Immobilized strains had lower efficiency in both GLU uptake and conversion compared to free cells, probably due to the poor diffusion in alginate beads. The use of resting cells allowed further GABA production without the cultivation step, but cell activity was exhausted after three cycles of reutilization. Lvb. brevis LB12 is an excellent GABA producer, and AE cultivation can be exploited to improve the final cell density; however, the conditions for boosting GLU to GABA conversion and cell regeneration need to be further investigated.

Effective removal of the rotifer Brachionus calyciflorus from a Chlorella vulgaris microalgal culture by homogeneous solar photo-Fenton at neutral pH

In this study, a citrate-modified photo-Fenton process was successfully applied to decontaminate a Chlorella vulgaris microalgae culture spiked with the rotifer Brachionus calyciflorus (5 individuals mL−1). The applied treatment (1 mg L−1 Fe2+, 20 mg L−1 H2O2, 17.5 mg L−1 citric acid) had only moderate effects on viability and regrowth of the microalgae since, after a short post-treatment delay of a few days, they reached final cell densities similar to that obtained for microalgae cultures that were not spiked. The decontamination was effective as no regrowth of rotifers was observed in the microalgae cultures after treatment. The efficacy of the citrate-modified photo-Fenton treatment was also studied with a higher starting concentration of 20 rotifers mL−1 and was compared with a solar light/H2O2 treatment. Results show that both treatments had similar efficacies on the rotifer elimination, but that the citrate-modified photo-Fenton treatment had a lower negative impact on the regrowth of microalgae than the solar light/H2O2 treatment. However, when microalgae cultures were spiked with 20 rotifers mL−1, rotifers were only partially inactivated and post-treatment regrowth occurred, which highlights the importance to apply the photo-Fenton process at an early stage of a contamination to achieve full rotifer elimination. In any case, a contamination with 5 rotifers mL−1 is already a significant threat as numbers above 1000 rotifers mL−1 were reached after 14 days and caused the microalgae culture to fail. Overall, our treatment suggests that the citrate-modified solar photo-Fenton process is an environmentally friendly solution to support the maintenance of contaminant-free microalgal cultures.

Economic optimization of expression of soluble human epidermal growth factor in Escherichia coli.

Human epidermal growth factor (hEGF) has multiple biological functions, such as promoting cell proliferation, differentiation, and migration. In addition, it is a very expensive polypeptide with attractive market prospects. However, the production of hEGF needs for high cost to manufacture polypeptide demands reinvestigations of process conditions so as to enhance economic benefits. Improving the expression of soluble hEGF is the fundamental method to reduce the cost. In this study, a non-extracellular engineered strain of expressed hEGF was constructed, using plasmid pET-22b(+) in Escherichia coli. Preliminary fermentation and high cell density cultivation were carried out in shake flasks and in a 5 L bioreactor, respectively. A high yield of 98 ± 10mg/L of soluble hEGF and a dry cell weight (DCW) of 6.98 ± 0.3g/L were achieved in shake flasks. Then, fermentation conditions were optimized for large-scale production, while taking into consideration the expensive equipment required for cooling and conforming to industrial standards. A yield of 285 ± 10mg/L of soluble hEGF, a final cell density of 57.4 ± 2g/L DCW (OD600 141.1 ± 4.9), and hEGF productivity of 14.3mg/L/h were obtained using a bioreactor at 32°C for 20h. The production method developed in this study for the biosynthesis of soluble hEGF is efficient and inexpensive.

Evaluating the microbial growth kinetics and artificial gastric digestion survival of a novel Pichia kudriavzevii FOL-04

Present study aims to explore Pichia kudriavzevii FOL-04 (FOL-04)’s: i) survival against artificial gastric juice (AGJ) and artificial bile juice (ABJ), ii) growth kinetics in shake flask (SF) and fed-batch trials (FBT). Survival of FOL-04 as measured by relative cell density (RCD) against AGJ and ABJ was screened at four different pH-levels (control, 3, 2, 1.5) and ox-bile concentrations (control, 0.2%, 1%, 2%), respectively. Growth kinetics was calculated by periodic measurement of OD600 in SF (225 rpm, 30°C) or in FBT using exponential feeding regimen where pH, dissolved-oxygen and temperature were controlled at 5.5, 21%, and 30°C, respectively. The doubling-time, maximum specific growth rate, and final cell densities achieved for SF and FBT were 81.7min, 1.67, 11.79 and 170.4 min, 4.75, 37.95, respectively. RCDs calculated were similar for pH=3 and control vs both were significantly higher(p&amp;lt;0.05) than pH=1.5 and 2 with the latter two pH-levels were not significantly different(p&amp;gt;0.05). RCDs were similar across control, 0.2%, and 1% ox-bile levels(p&amp;gt;0.05). However, 2% ox-bile yielded significantly lower RCD (p&amp;lt;0.05) compared to all except 1%. FOL-04 is a potential probiotic candidate showing robustness against AGJ and ABJ and remarkable biomass increase was achieved when grown under FBT which could pave the way for developing a yeast-based probiotic using this strain.

N-1 Perfusion Platform Development Using a Capacitance Probe for Biomanufacturing.

Fed-batch process intensification with a significantly shorter culture duration or higher titer for monoclonal antibody (mAb) production by Chinese hamster ovary (CHO) cells can be achieved by implementing perfusion operation at the N-1 stage for biomanufacturing. N-1 perfusion seed with much higher final viable cell density (VCD) than a conventional N-1 batch seed can be used to significantly increase the inoculation VCD for the subsequent fed-batch production (referred as N stage), which results in a shorter cell growth phase, higher peak VCD, or higher titer. In this report, we incorporated a process analytical technology (PAT) tool into our N-1 perfusion platform, using an in-line capacitance probe to automatically adjust the perfusion rate based on real-time VCD measurements. The capacitance measurements correlated linearly with the offline VCD at all cell densities tested (i.e., up to 130 × 106 cells/mL). Online control of the perfusion rate via the cell-specific perfusion rate (CSPR) decreased media usage by approximately 25% when compared with a platform volume-specific perfusion rate approach and did not lead to any detrimental effects on cell growth. This PAT tool was applied to six mAbs, and a platform CSPR of 0.04 nL/cell/day was selected, which enabled rapid growth and maintenance of high viabilities for four of six cell lines. In addition, small-scale capacitance data were used in the scaling-up of N-1 perfusion processes in the pilot plant and in the GMP manufacturing suite. Implementing a platform approach based on capacitance measurements to control perfusion rates led to efficient process development of perfusion N-1 for supporting high-density CHO cell cultures for the fed-batch process intensification.

Soft-sensors application for automated feeding control in high-throughput mammalian cell cultures.

The ever-increasing demand for biopharmaceuticals has created the need for improving the overall productivity of culture processes. One such operational concept that is considered is fed-batch operations as opposed to batch operations. However, optimal fed-batch operations require complete knowledge of the cell culture to optimize the culture conditions and the nutrients feeding. For example, when using high-throughput small-scale bioreactors to test multiple clones that do not behave the same, depletion or overfeeding of some key components can occur if the feeding strategy is not individually optimized. Over the recent years, various solutions for real-time measuring of the main cell culture metabolites have been proposed. Still, the complexity in the implementation of these techniques has limited their use. Soft-sensors present an opportunity to overcome these limitations by indirectly estimating these variables in real-time. This manuscript details the development of a new soft-sensor-based fed-batch strategy to maintain substrate concentration (glucose and glutamine) at optimal levels in small-scale multiparallel Chinese Hamster Ovary Cells cultures. Two alternatives to the standard feeding strategy were tested: an OUR soft-sensor-based strategy for glucose and glutamine (Strategy 1) and a dual OUR for glutamine and CO2 /alkali addition for glucose soft-sensor strategy (Strategy 2). The results demonstrated the applicability of the OUR soft-sensor-based strategy to optimize glucose and glutamine feedings, which yielded a 21% increase in final viable cell density (VCD) and a 31% in erythropoietin titer compared with the reference one. However, CO2 /alkali addition soft-sensor suffered from insufficient data to relate alkali addition with glucose consumption. As a result, the culture was overfed with glucose resulting in a 4% increase on final VCD, but a 9% decrease in final titer compared with the Reference Strategy.

Role of substrate availability in the growth of Campylobacter co-cultured with extended spectrum beta-lactamase-producing Escherichia coli in Bolton broth

It is well-established that Extended-spectrum beta-lactamase-producing (ESBL-) Escherichia coli challenge reliable detection of campylobacters during enrichment in Bolton broth (BB) following ISO 10272-1:2017. The overgrowth of Campylobacter by ESBL-E. coli in the enrichment medium BB can lead to false-negative detection outcomes, but the cause for the growth suppression is yet unknown. A plausible reason could be the competition-induced lack of certain growth substrates. Therefore, this study aimed to investigate whether campylobacters and ESBL-E. coli compete for the same medium components and whether this is the cause for the observed growth repression. The availability of possible growth substrates in BB was determined and changes in their extracellular concentration were measured over time during mono-culture enrichment of C. jejuni, C. coli or ESBL-E. coli as well as in co-culture enrichments of campylobacters and ESBL-E. coli. Comparative analysis showed lactate and fumarate utilization by C. jejuni and C. coli exclusively, whereas ESBL-E. coli rapidly consumed asparagine, glutamine/arginine, lysine, threonine, tryptophan, pyruvate, glycerol, cellobiose, and glucose. Both campylobacters and ESBL-E. coli utilized aspartate, serine, formate, a-ketoglutarate and malate. Trends in compound utilization were similar for C. jejuni and C. coli and trends in compound utilization were rather comparable during enrichment of reference and freeze-stressed campylobacters. Since final cell densities of C. jejuni and C. coli in co-cultures were not enhanced by the addition of surplus l-serine and final cell densities were similar in fresh and spent medium, growth suppression seems not to be caused by a lack of substrates or production of inhibitory compounds. We hypothesized that oxygen availability was limiting growth in co-cultures. Higher oxygen availability increased the competitive fitness of C. jejuni 81-176 in co-culture with ESBL-E. coli in duplicate experiments, as cell concentrations in stationary phase were similar to those without competition. This could indicate the critical role of oxygen availability during the growth of Campylobacter and offers potential for further improvement of Campylobacter spp. enrichment efficacy.

Potential of Acidithiobacillus ferrooxidans to Grow on and Bioleach Metals from Mars and Lunar Regolith Simulants under Simulated Microgravity Conditions.

The biomining microbes which extract metals from ores that have been applied in mining processes worldwide hold potential for harnessing space resources. Their cell growth and ability to extract metals from extraterrestrial minerals under microgravity environments, however, remains largely unknown. The present study used the model biomining bacterium Acidithiobacillus ferrooxidans to extract metals from lunar and Martian regolith simulants cultivated in a rotating clinostat with matched controls grown under the influence of terrestrial gravity. Analyses included assessments of final cell count, size, morphology, and soluble metal concentrations. Under Earth gravity, with the addition of Fe3+ and H2/CO2, A. ferrooxidans grew in the presence of regolith simulants to a final cell density comparable to controls without regoliths. The simulated microgravity appeared to enable cells to grow to a higher cell density in the presence of lunar regolith simulants. Clinostat cultures of A. ferrooxidans solubilised higher amounts of Si, Mn and Mg from lunar and Martian regolith simulants than abiotic controls. Electron microscopy observations revealed that microgravity stimulated the biosynthesis of intracellular nanoparticles (most likely magnetite) in anaerobically grown A. ferrooxidans cells. These results suggested that A. ferrooxidans has the potential for metal bioleaching and the production of useful nanoparticles in space.

Inhibitor tolerance and bioethanol fermentability of levoglucosan-utilizing Escherichia coli were enhanced by overexpression of stress-responsive gene ycfR: The proteomics-guided metabolic engineering

Pretreatment of lignocellulosic biomass is crucial for the release of biofermentable sugars for biofuels production, which could greatly alleviate the burgeoning environment and energy crisis caused by the massive usage of traditional fossil fuels. Pyrolysis is a cost-saving pretreatment process that can readily decompose biomass into levoglucosan, a promising anhydrosugar; however, many undesired toxic compounds inhibitory to downstream microbial fermentation are also generated during the pyrolysis, immensely impeding the bioconversion of levoglucosan-containing pyrolysate. Here, we took the first insight into the proteomic responses of a levoglucosan-utilizing and ethanol-producing Escherichia coli to three representative biomass-derived inhibitors, identifying large amounts of differentially expressed proteins (DEPs) that could guide the downstream metabolic engineering for the development of inhibitor-resistant strains. Fifteen up- and eight down-regulated DEPs were further identified as the biomarker stress-responsive proteins candidate for cellular tolerance to multiple inhibitors. Among these biomarker proteins, YcfR exhibiting the highest expression fold-change level was chosen as the target of overexpression to validate proteomics results and develop robust strains with enhanced inhibitor tolerance and fermentation performance. Finally, based on four plasmid-borne genes encoding the levoglucosan kinase, pyruvate decarboxylase, alcohol dehydrogenase, and protein YcfR, a new recombinant strain E. coli LGE-ycfR was successfully created, showing much higher acetic acid-, furfural-, and phenol-tolerance levels compared to the control without overexpression of ycfR. The specific growth rate, final cell density, ethanol concentration, ethanol productivity, and levoglucosan consumption rate of the recombinant were also remarkably improved. From the proteomics-guided metabolic engineering and phenotypic observations, we for the first time corroborated that YcfR is a stress-induced protein responsive to multiple biomass-derived inhibitors, and also developed an inhibitors-resistant strain that could produce bioethanol from levoglucosan in the presence of inhibitors of relatively high concentration. The newly developed E. coli LGE-ycfR strain that could eliminate the commonly-used costly detoxicification processes, is of great potential for the in situ cost-effective bioethanol production from the biomass-derived pyrolytic substrates.