Fed-batch Research Articles

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.

Efficient production of itaconic acid from the single-carbon substrate methanol with engineered Komagataella phaffii

BackgroundAmidst the escalating carbon dioxide levels resulting from fossil fuel consumption, there is a pressing need for sustainable, bio-based alternatives to underpin future global economies. Single-carbon feedstocks, derived from CO2, represent promising substrates for biotechnological applications. Especially, methanol is gaining prominence for bio-production of commodity chemicals.ResultsIn this study, we show the potential of Komagataella phaffii as a production platform for itaconic acid using methanol as the carbon source. Successful integration of heterologous genes from Aspergillus terreus (cadA, mttA and mfsA) alongside fine-tuning of the mfsA gene expression, led to promising initial itaconic acid titers of 28 g·L−1 after 5 days of fed-batch cultivation. Through the combined efforts of process optimization and strain engineering strategies, we further boosted the itaconic acid production reaching titers of 55 g·L−1 after less than 5 days of methanol feed, while increasing the product yield on methanol from 0.06 g·g−1 to 0.24 g·g−1.ConclusionOur results highlight the potential of K. phaffii as a methanol-based platform organism for sustainable biochemical production.

From yeast screening for suitability as single cell protein to fed-batch cultures.

Fed-batch cultures have rarely been used in single cell protein (SCP) research. This work evaluated multiple yeast species for suitability as SCP cultivated using glucose- and sucrose-based substrate and performed in-depth studies of fed-batch SCP cultivation kinetics for selected yeasts, including determination of specific crude nitrogen-to-protein conversion factors. SCP was cultivated using fully synthetic media in flask batch or bioreactor fed-batch cultures. Crude nitrogen and nucleic acid content were determined using the Dumas method and fluorescence assay kits, respectively. C. utilis compared favorably to other yeasts in flask batch cultures in terms of process yield (0.52 ± 0.01 gxgs-1) and crude nitrogen content (10.0 ± 0.5 and 9.9 ± 0.5%CDW for glucose and sucrose, respectively). This is the first time biomass composition data was reported for SCP cultivated in fed-batch mode. C. utilis crude nitrogen content was consistent across the tested conditions (protein content stabilized around 50%CDW in fed-batch), while that of the benchmark yeast S. cerevisiae was higher in batch cultures and at the beginning of fed-batch relative to the end (protein content decreased over time and stabilized around 43%CDW). Total nucleic acid content of the yeasts was similar (6.8%CDW and 6.3%CDW, for C. utilis and S. cerevisiae, respectively), with crude nitrogen-to-protein conversion factors of 4.97 and 5.80. This study demonstrated the suitability of C. utilis as SCP, notably the robustness of its crude nitrogen content (as an indicator of protein content) across batch and fed-batch conditions, compared to that of the benchmark yeast S. cerevisiae.

Optimization of the Process of Chinese Hamster Ovary (CHO) Cell Fed-Batch Culture to Stabilize Monoclonal Antibody Production and Overall Quality: Effect of pH Control Strategies

Optimization of the Process of Chinese Hamster Ovary (CHO) Cell Fed-Batch Culture to Stabilize Monoclonal Antibody Production and Overall Quality: Effect of pH Control Strategies

Propionate production by Bacteroidia gut bacteria and its dependence on substrate concentrations differs among species

BackgroundPropionate is a food preservative and platform chemical, but no biological process competes with current petrochemical production routes yet. Although propionate production has been described for gut bacteria of the class Bacteroidia, which also carry great capacity for the degradation of plant polymers, knowledge on propionate yields and productivities across species is scarce. This study aims to compare propionate production from glucose within Bacteroidia and characterize good propionate producers among this group.ResultsWe collected published information on propionate producing Bacteroidia, and selected ten species to be further examined. These species were grown under defined conditions to compare their product formation. While propionate, acetate, succinate, lactate and formate were produced, the product ratios varied greatly among the species. The two species with highest propionate yield, B. propionicifaciens (0.39 gpro/ggluc) and B. graminisolvens (0.25 gpro/ggluc), were further examined. Product formation and growth behavior differed significantly during CO2-limited growth and in resting cells experiments, as only B. graminisolvens depended on external-added NaHCO3, while their genome sequences only revealed few differences in the major catabolic pathways. Carbon mass and electron balances in experiments with resting cells were closed under the assumption that the oxidative pentose pathway was utilized for glucose oxidation next to glycolysis in B. graminisolvens. Finally, during pH-controlled fed-batch cultivation B. propionicifaciens and B. graminisolvens grew up to cell densities (OD600) of 8.1 and 9.8, and produced 119 mM and 33 mM of propionate from 130 and 105 mM glucose, respectively. A significant production of other acids, particularly lactate (25 mM), was observed in B. graminisolvens only.ConclusionsWe obtained the first broad overview and comparison of propionate production in Bacteroidia strains. A closer look at two species with comparably high propionate yields, showed significant differences in their physiology. Further studies may reveal the molecular basis for high propionate yields in Bacteroidia, paving the road towards their biotechnological application for conversion of biomass-derived sugars to propionate.

Sustainable production of yeast oil from rice straw hydrolysate by Pseudozyma parantarctica through fed-batch cultivation for bio-polyurethane foam formation

Rice straw serves as an alternative feedstock for yeast oil (YO) production and bio-polyurethane (BPU) foam formation. This study optimized yeast growth and YO production by Pseudozyma parantarctica, utilizing rice straw hydrolysate (RSH) as the primary carbon source. Fed-batch strategies were explored to enhance YO production efficiency and its conversion into BPU foam. The optimized conditions proved highly effective in yielding biomass and YO from RSH-OPM. The fed-batch approach significantly improved biomass, YO production, and oil content to 7.35 ± 0.18 g/L, 3.77 ± 0.09 g/L, and 51.21 ± 0.15 % (w/w), respectively. The main fatty acid (FA) compositions of YO consisted of C:16 to C:18 FAs for over 92 %. RSH-based YO was successfully transformed into both rigid and semi-rigid BPU foams. This research demonstrated the feasibility and potential of using RSH as a low-cost and renewable carbon source for YO production and BPU foam formation.

Metabolomic characterization of monoclonal antibody-producing Chinese hamster lung (CHL)-YN cells in glucose-controlled serum-free fed-batch operation.

The fast-growing Chinese hamster lung (CHL)-YN cell line was recently developed for monoclonal antibody production. In this study, we applied a serum-free fed-batch cultivation process to immunoglobulin (Ig)G1-producing CHL-YN cells, which were then used to design a dynamic glucose supply system to stabilize the extracellular glucose concentration based on glucose consumption. Glucose consumption of the cultures rapidly oscillated following three phases of glutamine metabolism: consumption, production, and re-consumption. Use of the dynamic glucose supply prolonged the viability of the CHL-YN-IgG1 cell cultures and increased IgG1 production. Liquid chromatography with tandem mass spectrometry-based target metabolomics analysis of the extracellular metabolites during the first glutamine shift was conducted to search for depleted compounds. The results suggest that the levels of four amino acids, namely arginine, aspartate, methionine, and serine, were sharply decreased in CHL-YN cells during glutamine production. Supporting evidence from metabolic and gene expression analyses also suggest that CHL-YN cells acquired ornithine- and cystathionine-production abilities that differed from those in Chinese hamster ovary-K1 cells, potentially leading to proline and cysteine biosynthesis.

High-Yield Production of Dihydroxyacetone from Crude Glycerol in Fed-Batch Cultures of Gluconobacter oxydans.

The strain Gluconobacter oxydans LMG 1385 was used for the bioconversion of crude glycerol to dihydroxyacetone. The suitability of fed-batch cultures for the production of dihydroxyacetone was determined, and the influence of the pH of the culture medium and the initial concentration of glycerol on maximizing the concentration of dihydroxyacetone and on the yield and speed of obtaining dihydroxyacetone by bioconversion was examined. The feeding strategy of the substrate (crude glycerol) during the process was based on measuring the dissolved oxygen tension of the culture medium. The highest concentration of dihydroxyacetone PK = 175.8 g·L-1 and the highest yield YP/Sw = 94.3% were obtained when the initial concentration of crude glycerol was S0 = 70.0 g·L-1 and the pH of the substrate was maintained during the process at level 5.0.

Metabolic engineering of CHO cells towards cysteine prototrophy and systems analysis of the ensuing phenotype

Chinese hamster ovary (CHO) cells require cysteine for growth and productivity in fed-batch cultures. In intensified processes, supplementation of cysteine at high concentrations is a challenge due to its limited solubility and instability in solution. Methionine can be converted to cysteine (CYS) but key enzymes, cystathionine beta-synthase (Cbs) and cystathionine gamma-lyase (Cth), are not active in CHO cells resulting in accumulation of an intermediate, homocysteine (HCY), in cell culture milieu. In this study, Cbs and Cth were overexpressed in CHO cells to confer cysteine prototrophy, i.e., the ability to grow in a cysteine free environment. These pools (CbCt) needed homocysteine and beta-mercaptoethanol (βME) to grow in CYS-free medium. To increase intracellular homocysteine levels, Gnmt was overexpressed in CbCt pools. The resultant cell pools (GnCbCt), post adaptation in CYS-free medium with decreasing residual HCY and βME levels, were able to proliferate in the HCY-free, βME-free and CYS-free environment. Interestingly, CbCt pools were also able to be adapted to grow in HCY-free and CYS-free conditions, albeit at significantly higher doubling times than GnCbCt cells, but couldn't completely adapt to βME-free conditions. Further, single cell clones derived from the GnCbCt cell pool had a wide range in expression levels of Cbs, Cth and Gnmt and, when cultivated in CYS-free fed-batch conditions, performed similarly to the wild type (WT) cell line cultivated in CYS supplemented fed-batch culture. Intracellular metabolomic analysis showed that HCY and glutathione (GSH) levels were lower in the CbCt pool in CYS-free conditions but were restored closer to WT levels in the GnCbCt cells cultivated in CYS-free conditions. Transcriptomic analysis showed that GnCbCt cells upregulated several genes encoding transporters as well as methionine catabolism and transsulfuration pathway enzymes that support these cells to biosynthesize cysteine effectively. Further, ‘omics analysis suggested CbCt pool was under ferroptotic stress in CYS-free conditions, which, when inhibited, enhanced the growth and viability of these cells in CYS-free conditions.

RNASeq highlights ATF6 pathway regulators for CHO cell engineering with different impacts of ATF6β and WFS1 knockdown on fed-batch production of IgG1

Secretion levels required of industrial Chinese hamster ovary (CHO) cell lines can challenge endoplasmic reticulum (ER) homeostasis, and ER stress caused by accumulation of misfolded proteins can be a bottleneck in biomanufacturing. The unfolded protein response (UPR) is initiated to restore homeostasis in response to ER stress, and optimization of the UPR can improve CHO cell production of therapeutic proteins. We compared the fed-batch growth, production characteristics, and transcriptomic response of an immunoglobulin G1 (IgG1) producer to its parental, non-producing host cell line. We conducted differential gene expression analysis using high throughput RNA sequencing (RNASeq) and quantitative polymerase chain reaction (qPCR) to study the ER stress response of each cell line during fed-batch culture. The UPR was activated in the IgG1 producer compared to the host cell line and our analysis of differential expression profiles indicated transient upregulation of ATF6α target mRNAs in the IgG1 producer, suggesting two upstream regulators of the ATF6 arm of the UPR, ATF6β and WFS1, are rational engineering targets. Although both ATF6β and WFS1 have been reported to negatively regulate ATF6α, this study shows knockdown of either target elicits different effects in an IgG1-producing CHO cell line. Stable knockdown of ATF6β decreased cell growth without decreasing titer; however, knockdown of WFS1 decreased titer without affecting growth. Relative expression measured by qPCR indicated no direct relationship between ATF6β and WFS1 expression, but upregulation of WFS1 in one pool was correlated with decreased growth and upregulation of ER chaperone mRNAs. While knockdown of WFS1 had negative impacts on UPR activation and product mRNA expression, knockdown of ATF6β improved the UPR specifically later in fed-batch leading to increased overall productivity.

Combinatorial Optimization Strategies for 3-Fucosyllactose Hyperproduction in Escherichia coli.

3-Fucosyllactose (3-FL), an important fucosylated human milk oligosaccharide in breast milk, offers numerous health benefits to infants. Previously, we metabolically engineered Escherichia coli BL21(DE3) for the in vivo biosynthesis of 3-FL. In this study, we initially optimized culture conditions to double 3-FL production. Competing pathway genes involved in in vivo guanosine 5'-diphosphate-fucose biosynthesis were subsequently inactivated to redirect fluxes toward 3-FL biosynthesis. Next, three promising transporters were evaluated using plasmid-based or chromosomally integrated expression to maximize extracellular 3-FL production. Additionally, through analysis of α1,3-fucosyltransferase (FutM2) structure, we identified Q126 residues as a highly mutable residue in the active site. After site-saturation mutation, the best-performing mutant, FutM2-Q126A, was obtained. Structural analysis and molecular dynamics simulations revealed that small residue replacement positively influenced helical structure generation. Finally, the best strain BD3-A produced 6.91 and 52.1 g/L of 3-FL in a shake-flask and fed-batch cultivations, respectively, highlighting its potential for large-scale industrial applications.

Ethanologenic fermentation by Parageobacillus thermoglucosidasius with continuous hot microbubble gas-stripping

The increased use of biofuels in place of fossil fuels is one strategy to support the transition to net-zero carbon emissions, particularly in transport applications. However, expansion of the use of 1st generation crops as feedstocks is unsustainable due to the conflict with food use. The use of the lignocellulosic fractions from plants and/or co-products from food production including food wastes could satisfy the demand for biofuels without affecting the use of land and the availability of food, but organisms which can readily ferment all the carbohydrates present in these feedstocks often suffer from more severe bioethanol inhibition effects than yeast. This paper demonstrates the potential of hot gas microbubbles to strip ethanol from a thermophilic fermentation process using Parageobacillus thermoglucosidasius TM333, thereby reducing product inhibition and allowing production to continue beyond the nominal toxic ethanol concentrations of ≤ 2% v/v. Using an experimental rig in which cells were grown in fed-batch cultures on sugars derived from waste bread, and the broth continuously cycled through a purpose-built microbubble stripping unit, it was shown that non/low-inhibitory dissolved ethanol concentrations could be maintained throughout, despite reaching productivities equivalent to 4.7% v/v dissolved ethanol. Ethanol recovered in the condensate was at a concentration appropriate for dewatering to be cost effective and not prohibitively energy intensive. This suggests that hot microbubble stripping could be a valuable technology for the continuous production of bioethanol from fermentation processes which suffer from product inhibition before reaching economically viable titres, which is typical of most thermophilic ethanologenic bacteria.

A production platform for disulfide-bonded peptides in the periplasm of Escherichia coli

BackgroundRecombinant peptide production in Escherichia coli provides a sustainable alternative to environmentally harmful and size-limited chemical synthesis. However, in-vivo production of disulfide-bonded peptides at high yields remains challenging, due to degradation by host proteases/peptidases and the necessity of translocation into the periplasmic space for disulfide bond formation.ResultsIn this study, we established an expression system for efficient and soluble production of disulfide-bonded peptides in the periplasm of E. coli. We chose model peptides with varying complexity (size, structure, number of disulfide bonds), namely parathyroid hormone 1–84, somatostatin 1–28, plectasin, and bovine pancreatic trypsin inhibitor (aprotinin). All peptides were expressed without and with the N-terminal, low molecular weight CASPON™ tag (4.1 kDa), with the expression cassette being integrated into the host genome. During BioLector™ cultivations at microliter scale, we found that most of our model peptides can only be sufficiently expressed in combination with the CASPON™ tag, otherwise expression was only weak or undetectable on SDS-PAGE. Undesired degradation by host proteases/peptidases was evident even with the CASPON™ tag. Therefore, we investigated whether degradation happened before or after translocation by expressing the peptides in combination with either a co- or post-translational signal sequence. Our results suggest that degradation predominantly happened after the translocation, as degradation fragments appeared to be identical independent of the signal sequence, and expression was not enhanced with the co-translational signal sequence. Lastly, we expressed all CASPON™-tagged peptides in two industry-relevant host strains during C-limited fed-batch cultivations in bioreactors. We found that the process performance was highly dependent on the peptide-host-combination. The titers that were reached varied between 0.6–2.6 g L−1, and exceeded previously published data in E. coli. Moreover, all peptides were shown by mass spectrometry to be expressed to completion, including full formation of disulfide bonds.ConclusionIn this work, we demonstrated the potential of the CASPON™ technology as a highly efficient platform for the production of soluble peptides in the periplasm of E. coli. The titers we show here are unprecedented whenever parathyroid hormone, somatostatin, plectasin or bovine pancreatic trypsin inhibitor were produced in E. coli, thus making our proposed upstream platform favorable over previously published approaches and chemical synthesis.

Multi-cell-line learning for the data-driven construction of mechanistic metabolic models.

Mammalian cells are commonly used as hosts in cell culture for biologics production in the pharmaceutical industry. Structured mechanistic models of metabolism have been used to capture complex cellular mechanisms that contribute to varying metabolic shifts in different cell lines. However, little research has focused on the impact of temporal changes in enzyme abundance and activity on the modeling of cell metabolism. In this work, we present a framework for constructing mechanistic models of metabolism that integrate growth-signaling control of enzyme activity and transcript dynamics. The proposed approach is applied to build models for three Chinese hamster ovary (CHO) cell lines using fed-batch culture data and time-series transcript profiles. Leveraging information from the transcriptome data, we develop a parameter estimation approach based on multi-cell-line (MCL) learning, which combines data sets from different cell lines and trains the individual cell-line models jointly to improve model accuracy. The computational results demonstrate the important role of growth signaling and transcript variability in metabolic models as well as the virtue of the MCL approach for constructing cell-line models with a limited amount of data. The resulting models exhibit a high level of accuracy in predicting distinct metabolic behaviors in the different cell lines; these models can potentially be used to accelerate the process and cell-line development for the biomanufacturing of new protein therapeutics.

Antibody glycan quality predicted from CHO cell culture media markers and machine learning

N-glycosylation can have a profound effect on the quality of mAb therapeutics. In biomanufacturing, one of the ways to influence N-glycosylation patterns is by altering the media used to grow mAb cell expression systems. Here, we explore the potential of machine learning (ML) to forecast the abundances of N-glycan types based on variables related to the growth media. The ML models exploit a dataset consisting of detailed glycomic characterisation of Anti-HER fed-batch bioreactor cell cultures measured daily under 12 different culture conditions, such as changes in levels of dissolved oxygen, pH, temperature, and the use of two different commercially available media. By performing spent media quantitation and subsequent calculation of pseudo cell consumption rates (termed media markers) as inputs to the ML model, we were able to demonstrate a small subset of media markers (18 selected out of 167 mass spectrometry peaks) in a Chinese Hamster Ovary (CHO) cell cultures are important to model N-glycan relative abundances (Regression - correlations between 0.80–0.92; Classification - AUC between 75.0–97.2). The performances suggest the ML models can infer N-glycan critical quality attributes from extracellular media as a proxy. Given its accuracy, we envisage its potential applications in biomaufactucuring, especially in areas of process development, downstream and upstream bioprocessing.

An event triggered control scheme for enhanced production of Escherichia coli and biomass concentration during fed-batch cultivation

Control of a bioprocess is a challenging task mainly due to the nonlinearity of the process, the complex nature of microorganisms, and variations in critical parameters such as temperature, pH, and agitator speed. Generally, the optimum values chosen for critical parameters during Escherichia coli (E.coli) K-12fed-batch fermentation are37 ᵒC for temperature, 7 for pH, and 35 % for Dissolved Oxygen (DO). The objective of this research is to enhance biomass concentration while minimizing energy consumption. To achieve this, an Event-Triggered Control (ETC) scheme based on feedback-feed forward control is proposed. The ETC system dynamically adjusts the substrate feed rate in response to variations in critical parameters. We compare the performance of classical Proportional Integral (PI) controllers and advanced Model Predictive Control (MPC) controllers in terms of bioprocess yield. Initially, the data are collected from a laboratory-scaled 3L bioreactor setup under fed-batch operating conditions, and data-driven models are developed using system identification techniques. Then, classical Proportional Integral (PI) and advanced Model Predictive Control (MPC) based feedback controllers are developed for controlling the yield of bioprocess by manipulating substrate flow rate, and their performances are compared. PI and MPC-based Event Triggered Feed Forward Controllers are designed to increase the yield and to suppress the effect of known disturbances due to critical parameters. Whenever there is a variation in the value of a critical parameter, it is considered an event, and ETC initiates a control action by manipulating the substrate feed rate. PI and MPC-based ETC controllers are developed in simulation, and their closed-loop performances are compared. It is observed that the Integral Square Error (ISE) is notably minimized to 4.668 for MPC with disturbance and 4.742 for MPC with Feed Forward Control. Similarly, the Integral Absolute Error (IAE) reduces to 2.453 for MPC with disturbance and 0.8124 for MPC with Feed Forward Control. The simulation results reveal that the MPC-based ETC control scheme enhances the biomass yield by 7 %, and this result is verified experimentally. This system dynamically adjusts the substrate feed rate in response to variations in critical parameters, which is a novel approach in the field of bioprocess control. Also, the proposed control schemes help reduce the frequency of communication between controller and actuator, which reduces power consumption.

D-Arabitol production by a high arabitol-producing yeast, Zygosaccharomyces sp. Gz-5 isolated from miso.

d-Arabitol, an alternative sweetener to sugar, has low calorie content, high sweetness, low glycemic index, and insulin resistance-improving ability. In this study, d-arabitol-producing yeast strains were isolated from various commercial types of miso, and strain Gz-5 was selected among these strains. Phylogenetic tree analysis of the internal transcribed spacer sequence revealed that strain Gz-5 was distinct from Zygosaccharomyces rouxii, a major fermenting yeast of miso. The strain, identified as Zygosaccharomyces sp. Gz-5, grew better than other Z. rouxii in 150g/L NaCl and produced 114g/L d-arabitol from 295g/L glucose in a batch culture for 8 days (0.386g/g-consumed glucose). In a fed-batch culture, the yeast produced 133g/L d-arabitol for 14 days, and the total d-arabitol amount increased by 1.75-fold. These results indicated that Zygosaccharomyces sp. Gz-5, a non-genetically modified strain, has excellent potential for the industrial production of d-arabitol.

Performance benchmark of different cell clones in discontinuous and continuous bioprocesses reveals critical impact of cellular diameter

Therapeutic proteins such as monoclonal antibodies are usually manufactured either in continuous processes such as steady-state perfusion or in discontinuous processes like fed-batch (FB). Thereby, both process formats have fundamentally different requirements for the utilized cell factories. This poses a problem as common cell line development programs are designed to select cell clones to perform well in FB cultivation. The aim of this study was to identify critical and easy to access cell line attributes for each process format and with that for the transfer of clones from fed-batch to perfusion. As a result, increased cell-volume specific productivity could be identified as beneficial within the FB not only impacting performance but as well diminishing media utilization and host-cell-proteins level. Within the perfusion, a stable cell diameter was identified as major beneficial characteristic, positively influencing cell viability, metabolite and glycan profile, as well as equipment utilization. Consistent with this, our data suggest that the inclusion of cell volume in the screening parameters is important for clone selection for both process formats and can lead to improved process design and robust cell line process transfer. Overall, this work gives valuable new insights in cell behaviour across discontinuous and continuous process formats to improve clone cell selection for both process strategies and streamline the process transfer from discontinuous towards continuous.

Cultivation of a Novel Strain of Chlorella vulgaris S2 under Phototrophic, Mixotrophic, and Heterotrophic Conditions, and Effects on Biomass Growth and Composition

Microalgal biomass is an excellent platform for producing food, feed, nutraceuticals, pharmaceuticals, and biofuels. This study aimed to investigate the effect of the trophic mode of cultivation (phototrophic, heterotrophic, and mixotrophic) on the growth and biomass composition of Chlorella vulgaris S2. The contents of lipids and carbohydrates, as well as the fatty acid composition of total lipids, were studied. The effects of the carbon-to-nitrogen ratio (C:N) and the organic carbon concentration of the growth media under mixotrophic and heterotrophic conditions were also investigated. The C:N ratio of 30 mol mol−1 favoured lipid synthesis, and the C:N ratio of 10 mol mol−1 favoured carbohydrate synthesis. Maximal lipid and biomass productivities (2.238 and 0.458 g L−1 d−1, respectively) were obtained under mixotrophic conditions at the C:N ratio of 50 mol mol−1 and glucose concentration of 50 g L−1. Fed-batch cultivation conducted in a stirrer tank bioreactor under heterotrophic growth conditions increased biomass (2.385 g L−1 d−1, respectively) and lipid (0.339 L−1 d−1) productivities ~50 and ~60 times compared to the fed-batch phototrophic cultivation, respectively. The trophic mode, growth phase, and growth medium composition significantly influenced the fatty acid composition. Under mixotrophic and heterotrophic growth conditions, lipid accumulation is associated with an increase in oleic acid (C18:1) content. Mixotrophically grown biomass of Chlorella vulgaris S2 under optimised conditions is a suitable source of lipids for biodiesel production.

Engineering Bacillus subtilis for highly efficient production of functional disaccharide lactulose from lactose

Bioconversion of lactose to functional lactose derivatives attracts increasing attention. Lactulose is an important high-value lactose derivative, which has been widely used in pharmaceutical, nutraceutical, and food industries. Lactulose can be enzymatically produced from lactose by cellobiose 2-epimerase (CEase). Several studies have already focused on the food-grade expression of CEase, but they are all aimed at the biosynthesis of epilactose. Herein, we reported for the first time the biosynthesis of lactulose using the recombinant food-grade Bacillus subtilis. Lactulose biosynthesis was optimized by varying lactulose-producing CEases and expression vectors. Caldicellulosiruptor saccharolyticus CEase and pP43NMK were determined to be the optimal CEase and expression vector. Fine-tuning of CEase expression was investigated by screening a beneficial N-terminal coding sequence. After fed-batch cultivation, the highest fermentation isomerization activity reached 11.6 U/mL. Lactulose was successfully produced by the broth of the engineered B. subtilis with a yield of 52.1 %.