Fed-batch Research Articles

Highly-efficient lipid production from hydrolysate of Radix paeoniae alba residue by oleaginous yeast Cutaneotrichosporon oleaginosum

Valorization of herbal extraction residues (HERs) into value-added products is pivotal for the sustainability of Chinese medicine industry. Here, seven different enzymatic hydrolysates of dilute acid pretreated HERs were evaluated for lipid production by Cutaneotrichosporon oleaginosum. Among them, the highest sugar yield via hydrolysis and the maximum lipid production were obtained from Radix paeoniae alba residue (RPAR). More interestingly, high proportion of sugar polymers was disintegrated into fermentable sugars during the pretreatment step, allowing a cheap non-enzymatic route for producing sugars from RPAR. A repeated dilute acid pretreatment gained a high sugar concentration of 241.6 g/L through reusing the pretreatment liquor (PL) for four times. Biomass, lipid concentration, and lipid content achieved 49.5 g/L, 35.7 g/L and 72.2 %, respectively, using fed-batch culture of PL. The biodiesel parameters indicated lipids produced from HERs were suitable for biodiesel production. This study offers a cost-effective way to upgrade the HERs waste into micro-biodiesel.

On-Line Reoptimization of Mammalian Fed-Batch Culture Using a Nonlinear Model Predictive Controller

On-Line Reoptimization of Mammalian Fed-Batch Culture Using a Nonlinear Model Predictive Controller

A two-phase approach optimizing productivity for a mAb-producing CHO cell culture process using dynamic response surface methodology models

Fed-batch cell culture processes are common in the biomanufacturing industry due to their ease of development and simplicity of execution in the cGMP environment. One challenge of fed-batch operation is that the physiochemical conditions within the bioreactor do not remain constant throughout a batch but change over time as the viable cell density changes. It is well accepted that fed-batch cultures are multi-phasic, divided into “growth, stationary, and death phases”. Yet, using empirical modeling methods such as response surface models (RSMs) for optimization is still common. While RSMs can identify local performance maxima, they are limited by the time-invariant factors (input parameters) they use. These factors do not change throughout the run. Dynamic RSM (DRSM) models predict the time-dependent impact of the process inputs allowing for the identification of optimized process operations that change with time. Starting with the data sets generated via a traditional Design of Experiments (DoE) design, the DRSM model successfully optimized the peak cell count during the growth phase of culture and then identified a second set of process parameters that optimized productivity during the stationary phase. The resulting process achieved a harvest titer improvement of 28% relative to the base process condition.

Kinetic and stoichiometric parameters in the fed-batch bioreactor production of poly(3-hydroxybutyrate) by Bacillus megaterium using different carbon sources.

This study investigates the effects of different strategies on poly(3-hydroxybutyrate)-P(3HB) production in a fed-batch bioreactor by Bacillus megaterium using candy industry effluent (CIE), sucrose, and rice parboiled water (RPW) as carbon sources. In biosynthesis, kinetic and stoichiometric parameters of substrate conversion into products and/or cells, productivity, instantaneous, and specific conversion rates were evaluated. The maximum concentration of P(3HB) was 4.00g.L-1 (77% of the total dry mass) in 42h of cultivation in minimal medium/RPW added with a carbon source based on CIE, demonstrating that the fed-batch provided an increase of approximately 22% in the polymer concentration and 32% in the overall productivity in relation to medium based on commercial sucrose. Fed-batch cultivation also had the advantage of avoiding the extra time required for inoculum preparation and sterilization of the bioreactor during the batch, which thereby increased the overall industrial importance of the process. Effluents from the candy, confectionery, and/or rice parboiling industries can be used as alternative substrates for P(3HB) production at a low cost.

Identification of Mispairing Omic Signatures in Chinese Hamster Ovary (CHO) Cells Producing a Tri-Specific Antibody.

Monoclonal antibody-based therapy has shown efficacy against cancer, autoimmune, infectious, and inflammatory diseases. Multispecific antibodies (MsAbs), including trispecifics (tsAbs), offer enhanced therapeutic potential by targeting different epitopes. However, when co-expressed from three or more different polypeptide chains, MsAb production can lead to incorrect chain assembly and co-production of mispaired species with impaired biological activity. Moreover, mispairing carries significant challenges for downstream purification, decreasing yields and increasing the cost of bioprocess development. In this study, quantitative transcriptomics and proteomics analyses were employed to investigate which signaling pathways correlated with low and high mispairing clone signatures. Gene and protein expression profiles of Chinese hamster ovary (CHO) clones producing an tsAb were analyzed in the exponential growth and stationary (tsAb production) phase of fed-batch culture. Functional analysis revealed activated endoplasmic reticulum stress in high mispairing clones in both culture phases, while low mispairing clones exhibited expression profiles indicative of activated protein translation, as well as higher endocytosis and target protein degradation, suggesting the clearance of unfolded proteins through ubiquitin-mediated mechanisms. In addition, through transcriptomic profiling, we identified a group of genes that have the potential to be used as a biomarker panel tool for identifying high mispairing levels in the early stages of bioprocess development.

A holistic approach for process intensification of nicotinamide mononucleotide production via high cell density cultivation under exponential feeding strategy

Nicotinamide mononucleotide (NMN) subsists in all living organisms and has drawn tremendous attention as a nutraceutical and pharmaceutical product for several diseases such as Alzheimer’s, cancer, aging, and vascular dysfunction. Here, NMN was produced intracellularly in a high cell density bioreactor using an engineered Escherichiacoli strain via exponential feeding of co-substrates. Fed-batch culture via exponential feeding of co-substrate (glucose) and continuous feeding of substrate (nicotinamide) were performed using different cumulative nicotinamide concentrations. The highest concentration of 19.3 g/L NMN with a dry cell weight of 117 g/L was acquired from a cumulative nicotinamide concentration of 7.2 g/L with a conversion of 98 % from nicotinamide in 28 h. Further, liquid chromatography-mass spectrometry analysis validated the NMN production. This approach will be beneficial in achieving simultaneously low cost and ensuring high quality and quantity of NMN production.

Ulva rigida Valorization into Poly(3-hydroxybutyrate), Organic Acids and Functional Ingredients.

Halomonas elongata 1H9T is a moderate halophilic strain able to produce poly(3-hydroxybutyrate) (P(3HB)), a biodegradable plastic, and gluconic acid, a valuable organic acid with wide industrial applications. In this work, the green alga Ulva rigida was used as platform to produce cultivation substrates for microbial conversion as well as functional ingredients, targeting its full valorization. The liquor obtained by autohydrolysis presented the highest concentration of oligosaccharides and protein, being an interesting feedstock to produce functional ingredients. The acid and/or enzymatic hydrolysis liquors are adequate as substrates for microbial processes. Shake flask assays with H. elongata revealed that the N-rich liquor produced after acidic treatment was the best suited for cell growth while the N-poor liquor produced by the enzymatic treatment of acid-pretreated algae residues produced the highest P(3HB) titers of 4.4 g/L. These hydrolysates were used in fed-batch cultivations as carbon and protein sources for the co-production of gluconic acid and polymer achieving titers of 123.2 g/L and 7.2 g/L, respectively. Besides gluconic acid, the Krebs cycle intermediate 2-oxoglutaric acid, also called alpha-ketoglutaric acid (KGA), was produced. Therefore, the co-production of P(3HB) and acids may be of considerable interest as an algal biorefinery valorization strategy.

Chaperonin co-expression and chemical modification enables production of active microbial transglutaminase from E. coli cytoplasm

Microbial transglutaminase (MTG) is a usable enzyme for biomacromolecule modification. In the present study, a “molecular chaperonin” strategy was developed to produce MTG in E. coli cytoplasm with high expression level and a “small molecule-mediated chemical modification” strategy was adopted to strip propeptide chaperonin efficiently during purification. Propeptide (Pro) was expressed separately as a chaperonin to facilitate MTG expression in E. coli cytoplasm with a yield up to 300 mg or about 9 kU from 1 L fed-batch culture. Furthermore, small molecular chemicals were applied to interfere the interaction between MTG and Pro. Chemical acetylation was identified as a suitable method to strip Pro resulting in pure MTG with high specific activity up to 49.6 U/mg. The purified acetylated MTG was characterized by MS analysis. The deconvoluted mass and Peptide Sequence Tags analysis confirmed acetylation on amino groups of MTG protein. Finally, the applications of obtained MTG were demonstrated via protein polymerization of bovine serum albumin and PEGylation of human interferon-α2b. Our method provides MTG with high purity and specific activity as well as unique merit with masked amino groups thus avoiding self-polymerization and cross-linking between MTG and substrates.

The Thermal Properties and Nutritional Value of Biomass of Oleaginous Yeast Rhodotorula sp. during Glucose Fed-Batch Cultivation in Medium with Waste Nitrogen

The biotechnological processing of oleaginous yeast biomass should be comprehensively managed using the zero-waste policy. This study focused on the biomass of the red yeast Rhodotorula obtained from a medium containing waste nitrogen. The cells accumulate lipids in intracellular lipid droplets; however, they are also rich in protein. Therefore, the nutritional value of lipid and protein, according to their fatty acid and amino acid composition, is a necessary step for practical application. For the very first time, this study focused on understanding the influence of temperature on powdered red yeast biomass to study components phase transition or chemical reactions by using DSC. Rhodotorula glutinis var. rubescens was cultivated in a glucose fed-batch in a potato wastewater medium, where the biomass yield was powdered, and lipid and fatty acid, protein, and amino acid compositions were determined. The DSC diagrams of red yeast biomass were characterized by two small and mild endothermic peaks, indicating the presence of fat and the presence of low molecular weight carbohydrates and a distinct peak associated with the presence of crystalline sugars. The nutritional quality of the lipid fraction as atherogenicity (0.223), thrombogenicity index (0.438), PUFA/SFA (0.24), and the n-6/n-3 ratio (3.275) was adequate for the recommendation and resulted from the fatty acid composition. The yeast protein was characterized by a high content of glutamic acid (99 mg/1 g of protein), and a value of essential amino acid index of protein suggested a superior amino acid composition compared to the FAO/WHO standard. Despite a high essential amino acid index (>120), yeast protein was characterized by a low content of Lys or Met.

Systematic sequence engineering enhances the induction strength of the glucose-regulated GTH1 promoter of Komagataella phaffii.

The promoter of the high-affinity glucose transporter Gth1 (PGTH1) is tightly repressed on glucose and glycerol surplus, and strongly induced in glucose-limitation, thus enabling regulated methanol-free production processes in the yeast production host Komagataella phaffii. To further improve this promoter, an intertwined approach of nucleotide diversification through random and rational engineering was pursued. Random mutagenesis and fluorescence activated cell sorting of PGTH1yielded five variants with enhanced induction strength. Reverse engineering of individual point mutations found in the improved variants identified two single point mutations with synergistic action. Sequential deletions revealed the key promoter segments for induction and repression properties, respectively. Combination of the single point mutations and the amplification of key promoter segments led to a library of novel promoter variants with up to 3-fold higher activity. Unexpectedly, the effect of gaining or losing a certain transcription factor binding site (TFBS) was highly dependent on its context within the promoter. Finally, the applicability of the novel promoter variants for biotechnological production was proven for the secretion of different recombinant model proteins in fed batch cultivation, where they clearly outperformed their ancestors. In addition to advancing the toolbox for recombinant protein production and metabolic engineering of K. phaffii, we discovered single nucleotide positions and correspondingly affected TFBS that distinguish between glycerol- and glucose-mediated repression of the native promoter.

Growth-coupled anaerobic production of isobutanol from glucose in minimal medium with Escherichia coli

BackgroundThe microbial production of isobutanol holds promise to become a sustainable alternative to fossil-based synthesis routes for this important chemical. Escherichia coli has been considered as one production host, however, due to redox imbalance, growth-coupled anaerobic production of isobutanol from glucose in E. coli is only possible if complex media additives or small amounts of oxygen are provided. These strategies have a negative impact on product yield, productivity, reproducibility, and production costs.ResultsIn this study, we propose a strategy based on acetate as co-substrate for resolving the redox imbalance. We constructed the E. coli background strain SB001 (ΔldhA ΔfrdA ΔpflB) with blocked pathways from glucose to alternative fermentation products but with an enabled pathway for acetate uptake and subsequent conversion to ethanol via acetyl-CoA. This strain, if equipped with the isobutanol production plasmid pIBA4, showed robust exponential growth (µ = 0.05 h−1) under anaerobic conditions in minimal glucose medium supplemented with small amounts of acetate. In small-scale batch cultivations, the strain reached a glucose uptake rate of 4.8 mmol gDW−1 h−1, a titer of 74 mM and 89% of the theoretical maximal isobutanol/glucose yield, while secreting only small amounts of ethanol synthesized from acetate. Furthermore, we show that the strain keeps a high metabolic activity also in a pulsed fed-batch bioreactor cultivation, even if cell growth is impaired by the accumulation of isobutanol in the medium.ConclusionsThis study showcases the beneficial utilization of acetate as a co-substrate and redox sink to facilitate growth-coupled production of isobutanol under anaerobic conditions. This approach holds potential for other applications with different production hosts and/or substrate–product combinations.

Optimized fed-batch cultivation of Rhodotorula toruloides in a bubble column bioreactor progressed the β-carotene production from corn steep liquor.

In this work, a new isolate yeast, namely Rhodotorula toruloides KP324973, was examined for β-carotene production from corn steep liquor (CSL) as a sole carbon source because CSL as the by-product of corn wet-milling process mainly enriched from the water-soluble carbohydrates. The studies were preliminary performed at the shaken flasks, and then developed at batch and fed-batch modes in a bubble column reactor (BCR). Application of the BCR improved the carotenogenesis of the cells in comparison with shaken flasks and the specific β-carotene production rate (Rp) and the yield of β-carotene production from the total reducing sugars (YP/TRS) reached 2.23 mg gcell-1 h-1 and 36.82 mg gTRS-1, respectively. Further studies were carried out to optimize the operational factors of the BCR for a fed-batch production by the response surface methodology. An optimal condition at a feed flow rate of 2.5 mL h-1, temperature 11.7°C, and initial pH of 6.1 obtained the highest Rp = 12.31 mg gcell-1 h-1 and YP/TRS = 97.18 mg gTRS-1.

Risk assessment for biopharmaceutical single-use manufacturing: A case study of upstream continuous processing

In the current transition to intensified upstream processing, the risks of adopting traditional single-use systems for high-titer, long-duration perfusion cultures, have thus far not been considered. This case study uses the Failure Modes and Effects Analysis (FMEA) method to evaluate the risks associated with implementing upstream single-use technology. The simulated model process was used to compare the risk level of single-use technology for a traditional fed-batch cell culture with that for perfusion culture, under the same annual protein production conditions. To provide a reasonable source of potential risk for FMEA, all single-use upstream operations for both fed-batch and perfusion processes were investigated using an analytical method developed to quantify the impact of process parameters and operating conditions on single-use system specifications and to ensure objectivity. Many of the risks and their levels, were similar in long-duration perfusion cultures and fed-batch cultures. However, differences were observed for high-risk components such as daily sampling and installation. The result of this analysis indicates that the reasons for risk are different for fed-batch cultures and perfusion cultures such as larger bioreactors in fed-batch and longer runs in perfusion, respectively. This risk assessment method could identify additional control measures and be part of a holistic contamination control strategy and help visualize their effectiveness.

Influencia de la variación de nutrientes en la alimentación sobre la acumulación de AbrB, esporulación y expresión de cry1Ac durante cultivos por lote alimentado de Bacillus thuringiensis

The biological control agent Bacillus thuringiensis (Bt) coordinates metabolic and gene regulation functions to enter to the differentiation process for the synthesis of spores and Cry toxin proteins. The master regulator for sporulation initiation is Spo0A, while AbrB regulator coordinate cellular functions during vegetative and transition growth. To understand how differentiation is modified during fed-batch cultures (FBC) of Bt under variable nutrient conditions, we conducted fermentations with two different nutrient concentration and feeding times using an industrial medium. Since sporulation efficiency has been lower in FBCs than in batch cultures, AbrB was monitored by Western blot during these cultures. Kinetic parameters were used to stablish timing of cell differentiation process, and fermentations were monitored on-line using impedance spectroscopy. AbrB was accumulated during the entire fed-batch culture, when a medium with the highest concentration of carbon and nitrogen sources (FBC1) were fed. Under these conditions, sporulation efficiency and cry1Ac expression were negatively affected, compared to the wild-type strain. Conversely, when the feeding concentration was only half of FBC1, AbrB accumulates in minor proportion, and sporulation efficiency and cry1Ac expression increased (FBC2). These results suggest that variable nutrient conditions can cause an imbalance in the accumulation of transition state regulators such as AbrB during FBCs, which can be detrimental to produce spores and Cry proteins, that are the main Bt products for biological control of agricultural pests.

Engineering Escherichia coli MG1655 for Highly Efficient Biosynthesis of 2'-Fucosyllactose by De Novo GDP-Fucose Pathway.

2'-Fucosyllactose (2'-FL), the most typical human milk oligosaccharide, is used as an additive in premium infant formula. Herein, we constructed two highly effective 2'-FL synthesis producers via a de novo GDP-fucose pathway from engineered Escherichia coli MG1655. First, lacZ and wcaJ, two competitive pathway genes, were disrupted to block the invalid consumption of lactose and GDP-fucose, respectively. Next, the lacY gene was strengthened by switching its native promoter to PJ23119. To enhance the supply of endogenous GDP-fucose, the promoters of gene clusters manC-manB and gmd-fcl were strengthened individually or in combination. Subsequently, chromosomal integration of a constitutive PJ23119 promoter-based BKHT expression cassette (PJ23119-BKHT) was performed in the arsB and recA loci. The most productive plasmid-based and plasmid-free strains produced 76.9 and 50.1 g/L 2'-FL by fed-batch cultivation, respectively. Neither of them generated difucosyl lactose nor 3-fucosyllactose as byproducts.

Economical production of Pichia pastoris single cell protein from methanol at industrial pilot scale

BackgroundMethanol, synthesized from CO2, is a potentially sustainable one-carbon (C1) resource for biomanufacturing. The use of methanol as a feedstock to produce single cell protein (SCP) has been investigated for decades as an alternative to alleviate the high global demand for animal-derived proteins. The methylotrophic yeast Pichia pastoris is an ideal host for methanol-based SCP synthesis due to its natural methanol assimilation ability. However, improving methanol utilization, tolerance to higher temperature, and the protein content of P. pastoris are also current challenges, which are of great significance to the economical industrial application using methanol as a feedstock for SCP production.ResultsIn the present work, adaptive laboratory evolution (ALE) has been employed to overcome the low methanol utilization efficiency and intolerance to a higher temperature of 33 °C in P. pastoris, associated with reduced carbon loss due to the lessened detoxification of intracellular formaldehyde through the dissimilation pathway and cell wall rearrangement to temperature stress resistance following long-term evolution as revealed by transcriptomic and phenotypic analysis. By strengthening nitrogen metabolism and impairing cell wall synthesis, metabolic engineering further increased protein content. Finally, the engineered strain via multi-strategy produced high levels of SCP from methanol in a pilot-scale fed-batch culture at 33 °C with a biomass of 63.37 g DCW/L, methanol conversion rate of 0.43 g DCW/g, and protein content of 0.506 g/g DCW. SCP obtained from P. pastoris contains a higher percentage of protein compared to conventional foods like soy, fish, meat, whole milk, and is a source of essential amino acids, including methionine, lysine, and branched-chain amino acids (BCAAs: valine, isoleucine, leucine).ConclusionsThis study clarified the unique mechanism of P. pastoris for efficient methanol utilization, higher temperature resistance, and high protein synthesis, providing a P. pastoris cell factory for SCP production with environmental, economic, and nutritional benefits.

Identification of optimal flow rate for culture media, cell density, and oxygen toward maximization of virus production in a fed-batch baculovirus-insect cell system.

In recent times, it has been realized that novel vaccines are required to combat emerging disease outbreaks, and faster optimization is required to respond to global vaccine demands. Although, fed-batch operations offer better productivity, experiment-based optimization of a new fed-batch process remains expensive and time-consuming. In this context, we propose a novel computational framework that can be used for process optimization and control of a fed-batch baculovirus-insect cell system. Since the baculovirus expression vector system (BEVS) is known to be widely used platforms for recombinant protein/vaccine production, we chose this system to demonstrate the identification of optimal profile. Toward this, first, we constructed a mathematical model that captures the time course of cell and virus growth in a baculovirus-insect cell system. Second, the proposed model was used for numerical analysis to determine the optimal operating profiles of control variables such as culture media, cell density, and oxygen based on a multiobjective optimal control formulation. Third, a detailed comparison between batch and fed-batch culture was perfromed along with a comparison between various alternatives of fed-batch operation. Finally, we demonstrate that a model-based quantification of controlled feed addition in fed-batch culture is capable of providing better productivity as compared to a batch culture. The proposed framework can be utilized for the estimation of optimal operating regions of different control variables to achieve maximum infected cell density and virus yield while minimizing the substrate/media, uninfected cell, and oxygen consumption.

High-cell-density cultivation of Vibrio natriegens in a low-chloride chemically defined medium

Vibrio natriegens is a halophilic bacterium with the fastest generation time of non-pathogenic bacteria reported so far. It therefore has high potential as a production strain for biotechnological production processes or other applications in biotechnology. Culture media for V. natriegens typically contain high sodium chloride concentrations. The corresponding high chloride concentrations can lead to corrosion processes on metal surfaces in bioreactors. Here we report the development of a low-chloride chemically defined medium for V. natriegens. Sodium chloride was completely replaced by the sodium salts disodium hydrogen phosphate, disodium sulfate, and sodium citrate, while keeping the total concentration of sodium ions constant. The use of citrate prevents the occurrence of precipitates, especially of ammonium magnesium phosphate. With this defined medium, high-cell-density fed-batch cultivations in laboratory-scale bioreactors using exponential feeding yielded biomass concentrations of more than 60 g L−1.Key pointsA defined medium for V. natriegens that only contains traces of chloride was developedCorrosion processes on metal surfaces in industrial bioreactors can thus be preventedHigh yields of biomass can be achieved in fed-batch cultivation with this medium

Computational redesign of taxane-10β-hydroxylase for de novo biosynthesis of a key paclitaxel intermediate.

Paclitaxel (Taxol®) is the most popular anticancer diterpenoid predominantly present in Taxus. The core skeleton of paclitaxel is highly modified, but researches on the cytochrome P450s involved in post-modification process remain exceedingly limited. Herein, the taxane-10β-hydroxylase (T10βH) from Taxus cuspidata, which is the third post-modification enzyme that catalyzes the conversion of taxadiene-5α-yl-acetate (T5OAc) to taxadiene-5α-yl-acetoxy-10β-ol (T10OH), was investigated in Escherichia coli by combining computation-assisted protein engineering and metabolic engineering. The variant of T10βH, M3 (I75F/L226K/S345V), exhibited a remarkable 9.5-fold increase in protein expression, accompanied by respective 1.3-fold and 2.1-fold improvements in turnover frequency (TOF) and total turnover number (TTN). Upon integration into the engineered strain, the variant M3 resulted in a substantial enhancement in T10OH production from 0.97 to 2.23mg/L. Ultimately, the titer of T10OH reached 3.89mg/L by fed-batch culture in a 5-L bioreactor, representing the highest level reported so far for the microbial de novo synthesis of this key paclitaxel intermediate. This study can serve as a valuable reference for further investigation of other P450s associated with the artificial biosynthesis of paclitaxel and other terpenoids. KEY POINTS: • The T10βH from T. cuspidata was expressed and engineered in E. coli unprecedentedly. • The expression and activity of T10βH were improved through protein engineering. • De novo biosynthesis of T10OH was achieved in E. coli with a titer of 3.89mg/L.

An Improved Cell Culture Process for Production of Omalizumab

The demand for biopharmaceutical products such as monoclonal antibodies (MAbs), fusion proteins, viral vaccines, and hormones is rapidly increasing. Mammalian cell cultures with ease of process operations are adopted to get the desired product. The challenges of low productivity and impurities in the desired product can be eliminated by optimizing the process parameters. The present study focuses on the optimization of temperature with other parameters to achieve improved titer and reduced heterogeneity in Omalizumab. An anti-IgE antibody expressed in mammalian cell culture. It is noticed that maintaining a specific temperature of 36.5° C ± 0.5 and pH 6.8± 0.1 during fed-batch cell culture provides improved quality (reduced galactosylation and reduced acidic variants) and quantity (improved titer) of the Omalizumab.
 Keywords: antibody, omalizumab, mammalian cells, acidic variants