Fed-batch Mode Research Articles

Efficient influenza A virus production in high cell density using the novel porcine suspension cell line PBG.PK2.1

Seasonal and pandemic influenza respiratory infections are still a major public health issue. Vaccination is the most efficient way to prevent influenza infection. One option to produce influenza vaccines is cell-culture based virus propagation. Different host cell lines, such as MDCK, Vero, AGE1.CR or PER.C6 cells have been shown to be a good substrate for influenza virus production. With respect to the ease of scale-up, suspension cells should be preferred over adherent cells. Ideally, they should replicate different influenza virus strains with high cell-specific yields. Evaluation of new cell lines and further development of processes is of considerable interest, as this increases the number of options regarding the design of manufacturing processes, flexibility of vaccine production and efficiency.Here, PBG.PK2.1, a new mammalian cell line that was developed by ProBioGen AG (Germany) for virus production is presented. The cells derived from immortal porcine kidney cells were previously adapted to growth in suspension in a chemically-defined medium. Influenza virus production was improved after virus adaptation to PBG.PK2.1 cells and optimization of infection conditions, namely multiplicity of infection and trypsin concentration. Hemagglutinin titers up to 3.24 log10(HA units/100 µL) were obtained in fed-batch mode in bioreactors (700 mL working volume). Evaluation of virus propagation in high cell density culture using a hollow-fiber based system (ATF2) demonstrated promising performance: Cell concentrations of up to 50 × 106 cells/mL with viabilities exceeding 95%, and a maximum HA titer of 3.93 log10(HA units/100 µL). Analysis of glycosylation of the viral HA antigen expressed showed clear differences compared to HA produced in MDCK or Vero cell lines. With an average cell-specific productivity of 5000 virions/cell, we believe that PBG.PK2.1 cells are a very promising candidate to be considered for next-generation influenza virus vaccine production.

High Conversion of D-Fructose into D-Allulose by Enzymes Coupling with an ATP Regeneration System.

D-Allulose is a rare monosaccharide that exists in extremely small quantities in nature, and it is also hard to prepare at a large scale via chemical or enzyme synthetic route due to low conversion and downstream separation complexity. Using D-psicose epimerase and L-rhamnulose kinase, a method enabling high conversion of D-allulose from D-fructose without the need for a tedious isomer separation step was established recently. However, this method requires expensive ATP to facilitate the reaction. In the present study, an ATP regenerate system was developed coupling with polyphosphate kinase. In our optimized reaction with purified enzymes, the conversion rate of 99% D-fructose was achieved at the concentrations of 2mM ATP, 5mM polyphosphate, 20mM D-fructose, and 20mMMg2+ when incubated at 50°C and at pH 7.5. ATP usage can be reduced to 10% of the theoretical amount compared to that without the ATP regeneration system. A fed-batch mode was also studied to minimize the inhibitory effect of polyphosphate. The biosynthetic system reported here offers a potential and promising platform for the conversion of D-fructose into D-allulose at reduced ATP cost.

Continuous operation, a realistic alternative to fed-batch fermentation for the production of recombinant lipase B from Candida antarctica under the constitutive promoter PGK in Pichia pastoris

In this work, continuous and fed-batch operational modes for producing the recombinant protein lipase B from Candida antarctica under the new constitutive promoter PGK in the cell factory Pichia pastoris was assessed. For this purpose, the influence of the specific growth rate (μ) was examined from 0.05 to 0.16 h−1 on various key bioprocess parameters.The overall biomass-to-substrate yield YX/S and specific substrate uptake rate (qs) were found to be independent of the particular operational mode. However, the μ-dependence of specific production rate (qp) differed with the cultivation strategy used. Thus, qp was linearly related in the continuous mode but suggested saturation in fed-batch cultures.The highest volumetric and specific productivity were obtained at the highest μlevels in the continuous mode, which provided volumetric and specific productivities (QP and Qe, respectively) roughly 1.5 and 3 times greater than the fed-batch mode. The continuous mode also proved more effective in the long rung; thus, CalB production after 6 weeks was estimated to be about 5.8 times greater than with the fed-batch mode.Continuous operation with the novel constitutive promoter PGK is thus a realistic alternative to the standard fed-batch cultivation at a high cell density typically used in industrial bioprocesses for heterologous protein production in Pichia pastoris.

Optimal feeding strategy of Cephalosporin C fermentation

Cephalosporin C is a β-lactam type of antibiotic produced from the fungus Acremonium chrysogenum through fermentation process, either by batch or fed-batch mode. Cephalosporin represents the bulk majority of antibiotic production due to its enhanced antibacterial spectrum against gram positive and gram negative bacterial strains relating to diseases and infections in the skin, respiratory system, and urinary tract. In the production process, it is crucial to maximize the yield of antibiotic produced, since the major costs of production come from the fermentation and recovery of the antibiotic. To address this issue, an effective feeding strategy of the culture producing the target antibiotic is very important to achieve high yield and avoid undesired production of other metabolites, which reduce the yield of desired metabolite. The latter can add extra cost to recovery and purification of the desired metabolite. This paper presents an optimal strategy for the fed-batch fermentation process producing Cephalosporin C (CPC) from the fungus A. chrysogenum by optimization of the substrate feeding flow rate. The preferred substrates are glucose and sucrose as the fungus possesses diauxic behaviour in the presence of the two carbon sources. It is shown that a two-step fed-batch feeding of glucose results in a significantly higher antibiotic production than a batch mode or single-step fed-batch feeding strategy.

Recovery of Lignosulfonates from Spent Sulfite Liquor Using Ceramic Hollow-Fiber Membranes.

Spent sulfite liquor is an abundant but currently less used wastewater stream from the pulp and paper industry. The recovery of lignin from this resource would provide an inexpensive raw material for the manufacture of fuels and fine chemicals. Here we investigated the suitability of ceramic hollow-fiber membranes for the concentration of spent sulfite liquor as an alternative to common membrane technologies. We tested three ceramic hollow-fiber membranes (3, 8, and 30 nm) in different membrane processes (fed-batch and total recycle mode) and compared their performance with the widely-used tubular membrane geometry. We also evaluated backflushing as a strategy to reduce membrane fouling during filtration. The juxtaposition of the two membrane geometries revealed that wall shear stress is the most important process parameter for the assessment of membrane performance according to permeate flux. The higher the wall shear stress, the higher the permeate flux. Due to the smaller inner diameter of the hollow-fiber membranes, higher wall shear stress can be achieved more easily. Backflushing had no effect on the permeate flux during the concentration experiments.

Polyhydroxyalkanoate production using waste vegetable oil and filtered digestate liquor of chicken manure

The production of polyhydroxyalkanoates (PHA) using digestate of chicken manure combined with waste sunflower oil as no-cost feedstocks in a multi-stage process was investigated. Using Cupriavidus necator H16 in combined culture media, a maximum PHA accumulation of 4.6 ± 0.2 g/L at 75.1 ± 1.4% of cell dry matter and a residual cell matter yield of 1.5 ± 0.1 g/L were obtained after 96 hr of cultivation (30 °C, 160 rpm, pH 7.0) in flask-based experiments. Manure was acidogenically fermented in a continuous stirring tank reactor in fed-batch mode. The bioreactor was operated at varying organic loading rates (OLR) and hydraulic retention times (HRT) ranging from 1–4 g volatile solids (VS)/L/d and 4–8 days, respectively. Optimal operation was observed at an OLR of 4 g VS/L/d and an HRT of 4 days. Analysis showed the presence of significant amounts of ammonia, potassium, magnesium, calcium, and trace elements, i.e. Fe, Cu, Ni, Mn, Co, Zn, Cr in the digestate. The micro-filtered digestate was utilized as a complex culture media base while waste oil served as an additional carbon source and supplemented for effective PHA production. The total volatile fatty acid content of digestate greatly affected the growth performance of the PHA-producing microorganism Cupriavidus necator H16.

Varied infeed inorganics and organics for the assimilation of aqueous petrochemical products in anoxic fed-batch reactors: Maximizing precipitation of S0

Feed sulfide (S2−) (0–1222 mg/L), phenol (500–1217 mg/L), diesel (0–700 mg/L), NH4+-N (0–450 mg/L) and NO3–-N (750–2000 mg/L) were varied in eight separate reactors (R1 to R8) operated in fed batch mode at constant hydraulic retention time (HRT: 1.25 day). Once a pollutant was varied, concentrations of others were kept constant. Maximum conversion of S2− to S0 occurred at the following influent concentrations: S2− 750 mg/L, phenol 750 mg/L, diesel 300 mg/L, NH4+-N 350 mg/L and NO3–-N 1000 mg/L. Elemental S0 production initially increased with increase in feed S2−, phenol and diesel and decreased at beyond feed concentrations of 750, 750 and 300 mg/L respectively. More than 350 mg/L of influent NH4+-N had detrimental effect on the oxidation of both organics and S2−. Conversion of S2− to SO42− was supported by higher load of NO3–-N. Gas produced mostly (80–90%) consisted of N2. Increase in suspended biomass was observed with increase in S2− load and attached biomass increased when organic load was increased.

Interaction between the production of ethanol and glycerol in fed-batch bioreactors.

During alcoholic fermentation, most of the substrates supplied to the yeasts are converted into ethanol and carbon dioxide generating energy for cell maintenance. However, some of these substrates end up being diverted to other metabolic pathways generating by-products reducing the yield in ethanol production. Glycerol is the most important by-product quantitatively, and its production during fermentation is associated to the production of ethanol. The present study was carried out at a full scale in an industrial fermentation plant applied to sugar cane industry with bioreactors operated in fed-batch mode. Varying levels of the operating factors feeding time, temperature, and concentration of yeast were used in order to verify the interaction between ethanol and glycerol in the fermentative kinetics and how these factors can be optimized to increase ethanol production with reduced carbon losses during the formation of other products. The results obtained indicated that glycerol production is linearly associated with ethanol production and that this correlation is influenced by the process conditions. Feeding time had a significant effect and was inversely proportional to the glycerol/ethanol production ratio. Therefore, it can be said that a moderate feeding rate can reduce the production of glycerol in relation to the ethanol produced reducing losses and increasing the fermentation yield.

Introducing substrate limitations to overcome catabolite repression in a protease producing Bacillus licheniformis strain using membrane‐based fed‐batch shake flasks

To overcome catabolite repression, industrial fermentation processes are usually operated in substrate-limited fed-batch mode. Therefore, the implementation of such an operating mode at small scale is crucial to maintain comparable process conditions. In this study, Bacillus licheniformis, a well-known producer of proteases, was cultivated with carbon (glucose)- and nitrogen (ammonium)-limited fed-batch conditions using the previously introduced membrane-based fed-batch shake flasks. A repression of protease production by glucose and ammonium was thus avoided and yields increased 1.5- and 2.1-fold relative to batch, respectively. An elevated feeding rate of glucose caused depletion of ammonium, which was recognizable within the oxygen transfer rate (OTR) signal measured with the Respiration Activity MOnitoring System (RAMOS). Ammonium limitation was prevented by feeding ammonium simultaneously with glucose. The OTR signal clearly indicated the initiation of the fed-batch phase and gave direct feedback on the nutrient release kinetics. Increased feeding rates of glucose and ammonium led to an elevated protease activity without affecting the protease yield (YP/Glu ). In addition to YP/Glu , protease yields were determined based on the metabolized amount of oxygen . The results showed that the protease production correlated with the amount of consumed glucose as well as with the amount of consumed oxygen. The membrane-based fed-batch shake flask in combination with the RAMOS device is a powerful combination to investigate the effect of substrate-limited fed-batch conditions.

CHO cell cultures in shake flasks and bioreactors present different host cell protein profiles in the supernatant

Several studies on the impact of cell culture parameters on the profile of host cell protein (HCP) impurities have been carried out in shake flasks. Herein, we explore how transferable the findings and conclusions of such investigations are to lab-scale bioreactors. Experiments were performed in both systems in fed-batch mode under physiological temperature and with a shift to mild hypothermia and the impact on key upstream performance indicators was quantified. Under both temperatures, bioreactors produced a richer HCP pool despite the overall concentration being similar at both scales and temperatures. The number of different HCPs detected in bioreactor supernatants was four times higher than that in flasks under physiological temperature and more than eight times higher under mild hypothermia. The origin of HCPs was also altered from mostly naturally secreted proteins in flasks to mainly intracellular proteins in bioreactors at the lower temperature. Although the number of species correlated with apoptotic cell density in bioreactors, this was not the case in flasks. Even though the level of HCP impurities and mAb/HCP concentration ratio were similar under all four conditions with an average of approximately 330 μg HCP/mL culture and 0.3 mg HCP/mg IgG4, respectively, the fact that culture method significantly affects the number of species present in the supernatant can have implications for downstream processing steps.

A multi-enzyme cascade reaction for the production of 6-hydroxyhexanoic acid.

Multi-enzyme cascade reactions capture the essence of nature's efficiency by increasing the productivity of a process. Here we describe one such three-enzyme cascade for the synthesis of 6-hydroxyhexanoic acid. Whole cells of Escherichia coli co-expressing an alcohol dehydrogenase and a Baeyer-Villiger monooxygenase (CHMO) for internal cofactor regeneration were used without the supply of external NADPH or NADP+. The product inhibition caused by the ε-caprolactone formed by the CHMO was overcome by the use of lipase CAL-B for in situ conversion into 6-hydroxyhexanoic acid. A stirred tank reactor under fed-batch mode was chosen for efficient catalysis. By using this setup, a product titre of >20 g L-1 was achieved in a 500 mL scale with an isolated yield of 81% 6-hydroxyhexanoic acid.

Viability of Using Glycerin as a Co-substrate in Anaerobic Digestion of Sugarcane Stillage (Vinasse): Effect of Diversified Operational Strategies

Vinasse, from sugar and ethanol production, stands out as one of the most problematic agroindustry wastes due to its high chemical oxygen demand, large production volume, and recalcitrant compounds. Therefore, the viability of using glycerin as a co-substrate in vinasse anaerobic digestion was tested, to increase process efficiency and biogas productivity. The effect of feeding strategy, influent concentration, cycle length, and temperature were assessed to optimize methane production. Glycerin (1.53% v/v) proved to be a good co-substrate since it increased the overall methane production in co-digestion assays. CH4 productivity enhanced exponentially as influent concentration increased, but when temperature was increased to 35°C, biogas production was impaired. The highest methane productivity and yield were achieved using fed-batch mode, at 30°C and at an organic loading rate of 10.1kgCODm-3day-1: 139.32molCH4m-3day-1, 13.86molCH4kgCODapplied, and 15.30molCH4kgCODremoved. Methane was predominantly produced through the hydrogenotrophic route. In order to treat all the vinasse produced by a mid-size sugar and ethanol plant, nine reactors with 7263.4m3 each would be needed. The energy generated by burning the biogas in boilers would reach approximately 92,000MWh per season and could save up to US$ 240,000.00 per month in diesel oil demand.

Process intensification for the production of rituximab by an inducible CHO cell line.

Mammalian-inducible expression systems are increasingly available and offer an attractive platform for the production of recombinant proteins. In this work, we have conducted process development for a cumate-inducible GS-CHO cell-line-expressing rituximab. To cope with the limitations encountered in batch when inducing at high cell densities, we have explored the use of fed-batch, sequential medium replacements, and continuous perfusion strategies applied during the pre-induction (growth) phase to enhance process performance in terms of product yield and quality. In shake flask, a fed-batch mode and a complete medium exchange at the time of induction were shown to significantly increase the integral of viable cell concentration and antibody titer compared to batch culture. Further enhancement of product yield was achieved by combining bolus concentrated feed additions with sequential medium replacement, but product galactosylation was reduced compared to fed-batch mode, as a result of the extended culture duration. In bioreactor, combining continuous perfusion of the basal medium with bolus daily feeding during the pre-induction period and harvesting earlier during the production phase is shown to provide a good trade-off between antibody titer and product galactosylation. Overall, our results demonstrate the importance of selecting a suitable operating mode and harvest time when carrying out high-cell-density induction to balance between culture productivity and product quality.

Intensification of a novel absorption-microalgae hybrid CO2 utilization process via fed-batch mode optimization

Ammonia-absorption-microalgae hybrid CO2 capture process presented the potential advantages in cost-effective CO2 capture and bioconversion. However, cultivation mode still needs to be intensified to reduce the negative influence of NH3 escape and its inhibition on microalgae growth. In the current work, three batch feeding modes of ammonium (i.e. NH4HCO3) for two Chlorella sp. (UTEX1602 and L38) were investigated. The experimental results indicated that initial NH4HCO3 deficiency (mode A) was beneficial for microalgae growth compared to the cultivation modes of uniform feeding (mode B) and decreasing dosage (mode C). Chlorella L38 presented a higher growth rate than UTEX1602, and might be more suitable for absorption-microalgae hybrid CO2 capture system. In detail, carbon utilization efficiency could achieve up to 76.8% in the absorption-microalgae hybrid process, which was two to three times higher than the conventional microalgae CO2 fixation processes. NH3 escape rate in absorption-microalgae hybrid process could be reduced to 24.2%, which was 18.8% lower than the conventional ammonia CO2 absorption process. Moreover, nitrogen and carbon in NH4HCO3 could be converted into biomass and value-added ingredients to realize resource recycling.

Biochemical characterization and biocatalytic application of a novel d-tagatose 3-epimerase from Sinorhizobium sp.

Sinorhizobium sp. d-tagatose 3-epimerase (sDTE) catalyzes the conversion of d-tagatose to d-sorbose. It also recognizes d-fructose as a substrate for d-allulose production. The optimal temperature and pH of the purified sDTE was 50 °C and 8.0, respectively. Based on the sDTE homologous model, Glu154, Asp187, Gln213, and Glu248, form a hydrogen bond network with the active-site Mn2+ and constitute the catalytic tetrad. The amino acid residues around O-1, -2, and -3 atoms of the substrates (d-tagatose/d-fructose) are strictly conserved and thus likely regulate the catalytic reaction. However, the residues at O-4, -5, and -6, being responsible for the substrate-binding, are different. In particular, Arg65 and Met9 were found to form a unique interaction with O-4 of d-fructose and d-tagatose. The whole cells with recombinant sDTE showed a higher bioconversion rate of 42.5% in a fed-batch bioconversion using d-fructose as a substrate, corresponding to a production of 476 g L−1d-allulose. These results suggest that sDTE is a potential industrial biocatalyst for the production of d-allulose in fed-batch mode.

Overproduction of lipopeptide biosurfactant by Aneurinibacillus thermoaerophilus HAK01 in various fed-batch modes under thermophilic conditions.

An efficient lipopeptide biosurfactant (BS) producer, Aneurinibacillus thermoaerophilus HAK01, was isolated from municipal landfill sites. The strain was able to produce about 4.9 g L−1 lipopeptide at a thermophilic temperature of 45 °C. After optimization of culture component concentrations using the response surface method, the main focus is to find the most appropriate fed-batch strategy to enhance lipopeptide production by the HAK01 strain. For this purpose, four fed-batch strategies including (a) pH-stat mode, (b) constant feeding rate strategy, (c) DO-stat mode, and (d) combined feeding strategy were designed. The production of BS was increased systematically from 4.9 g L−1 in batch mode to 5.9, 7.1, 8.8 and 11.2 g L−1 in each fed-batch mode, respectively. While poor results were obtained in the pH-stat mode, the DO-stat mode showed excellent results in the production of BS. The results of the study confirmed the importance of operational mode, oxygen supply and the kind of feeding strategy in BS production.

Принцип оперативного выбора лидерных клонов-продуцентов моноклональных антител при создании стабильных клеточных линий на основе CHO

Growth and productive characteristics of monoclonal cell lines based on CHO cells and producing a therapeutic protein have been monitored using the robot Ambr Tap Biosystems, which permitted to identify the leading line. Twenty four clones producing a recombinant monoclonal antibody were studied under the close to industrial conditions in a fed-batch culturing mode. The ambr®15 cell culture workstation controls 24 disposable mini bioreactors, and offers parallel processing and evaluation of multiple (24) experiments in an automated bench-top system. The volumetric productivity of 24 clones determined by ELISA was 120-450 mg /L. A protocol was shown to select a leader among producing clones for further research. producing clones, mini bioreactor; Ambr Tap Biosystems, fed-batch, monoclonal antibodies, CHO cell culture

Combinatorial impact of Sec signal peptides from Bacillus subtilis and bioprocess conditions on heterologous cutinase secretion by Corynebacterium glutamicum.

The impact of Sec signal peptides (SPs) from Bacillus subtilis in combination with isopropyl-β- d-1-thiogalactopyranoside concentration and feeding profile was investigated for heterologous protein secretion performance by Corynebacterium glutamicum using cutinase as model enzyme. Based on a comprehensive data set of about 150 bench-scale bioreactor cultivations in fed-batch mode and choosing the cutinase yield as objective, it was shown that relative secretion performance for bioprocesses remains very similar, irrespective of the applied SP enabling Sec-mediated cutinase secretion. However, to achieve the maximal absolute cutinase yield, careful adjustment of bioprocess conditions was found to be necessary. A model-based, two-step multiple regression approach resembled the collected data in a comprehensive way. The corresponding results suggest that the choice of the heterologous Sec SP and its interaction with the adjusted exponential feeding profile is highly relevant to maximize absolute cutinase yield in this study. For example, the impact of Sec SP is high at low growth rates and low at high growth rates. However, promising Sec SPs could be inferred from less complex batch cultivations. The extensive data were also evaluated in terms of cutinase productivity, highlighting the well-known trade-off between yield and productivity in bioprocess development in detail. Conclusively, only the right combination of target protein, Sec SP, and bioprocess conditions is the key to success.

Ethanol production from sugarcane bagasse: Use of different fermentation strategies to enhance an environmental-friendly process

Ethanol production by simultaneous saccharification and fermentation (SSF) using sugarcane bagasse as substrate was developed using batch and fed-batch mode. Acid, alkali, hydrothermal and hydrogen peroxide pretreatments to the sugarcane bagasse were tested. Experiments were carried out to optimize the enzyme load of cellulases and β-glucosidase. Four strains, two of Saccharomyces cerevisiae and two of Kluyveromyces marxianus yeast species were evaluate using SSF to produce ethanol. A kinetic study in bioreactor was carried out to optimize the SSF. The batch process was optimized using 1.0 g/L of inoculum, 15.0 FPU/g cellulose of cellulases and 6.0% of initial cellulose reaching 92.0% of theoretical ethanol yield after 18 h using the bagasse pretreate by acid-alkali and S. cerevisiae PE-2. The fed-batch process with enzyme load three times lower than that was used in batch process, obtained 88% of theoretical ethanol yield in 40 h. Therefore, the use of the lignocellulosic biomass (sugarcane bagasse) for producing a biofuel (ethanol) reduces the need for oil and is an environmental-friendly process.

Domestic wastewater treatment in a tubular microbial electrolysis cell with a membrane electrode assembly

High hydrogen production rate and energy recovery were accomplished in a tubular microbial electrolysis cell (MEC) equipped with a robust membrane electrode assembly (MEA). The current and the hydrogen production of non-flexible MEAs, simply fabricated by directly brushing a catalyst on a self-supporting tubular membrane, were compared with those of a typical MEA, where a cathode is physically combined with a membrane. Current of 34 ± 2 mA (1.79 ± 0.05 A/m2) and coulombic efficiency of 98.5 ± 1.0% were achieved in non-flexible MEAs, outperforming the typical MEA under fed-batch mode. The MEA, having a durable coating layer, also showed an enhanced hydrogen production rate and electric energy recovery with values of 0.18 ± 0.03 m3/m3-d and 151.9 ± 1.0%, respectively, even for low strength domestic wastewater (dWW) treatment in the continuous-flow mode. These outcomes were similarly maintained in the case of using seawater, which is a good candidate for an economical and environmentally suitable catholyte.