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Monoclonal antibodies (mAbs) used in biomedical research and therapeutic applications are primarily produced by Chinese hamster ovary (CHO) cells via fed-batch culture. The growing need for elevated quantities of biologics mandates the continual optimization of the mAb production process. The development of an effective process control method is indispensable for the production of specified mAbs by CHO cells. In this study, the effects of the pH control strategy on CHO cell fed-batch culture to produce an antibody (EA5) were first investigated in a 3 L bioreactor. The results indicate that controlling the culture pH at 7.2 during the fed-batch stage could produce a higher EA5 titer of 6.1 g/L with a lower Man5 ratio of 2.2% by day 14. Based on this, an optimized CHO cell fed-batch culture was conducted in a 15 L bioreactor to verify its effectiveness and stability. In this case, on day 14, an EA5 titer of 6.5 g/L was achieved with productivity of 0.46 g/L/day, which was 1.07-fold higher compared to that of the culture in the 3 L bioreactor. Furthermore, regarding the product quality, a monomer abundance of 96.0%, a main peak of 55.0%, and a Man5 proportion of 2.4% were maintained in the 15 L bioreactor. In addition, different cell clarification processes were evaluated using the CHO cell culture broth from the 3 L and 15 L bioreactors to further improve productivity and economic performance. Overall, this study provides some directions for process intensification and improving the quality of mAbs produced by CHO cells in the biopharmaceutical industry. [ABSTRACT FROM AUTHOR]

BACKGROUND: 1,3‐Propanediol (PDO) is a raw material/specialty chemical/additive in several industrial applications. A new wild‐type Clostridium strain BOH3 can utilize crude glycerol (CG) and produce PDO. This investigation reveals its potential for PDO production from CG. RESULTS: Initially, inoculum of strain BOH3 was cultured from reinforced clostridial medium (RCM) with glucose (medium‐A), and modified‐RCM with glycerol (medium‐B). While fermenting 20 g L−1 glycerol, the cells cultivated from medium‐A produced 5.75 g L−1 PDO (as major product) with 3.10 g L−1 butanol (byproduct), whereas the cells cultivated from medium‐B produced 11.01 g L−1 PDO with 2.50 g L−1 butyric acid and 1.01 g L−1 acetic acid, with no butanol produced. To enhance PDO production from the batch‐process, various parameters including pure glycerol/CG concentration (80 g L−1), inoculum age (15 h) and size (12.50%v/v) of cells from medium‐B, initial medium‐pH (pH 6.4) and incubation temperature (39 °C) were studied; the optimal values obtained are given in their respective parenthesis. Under optimal conditions, PDO production was 42.58–47.15 g L−1 (0.65–0.79 mol‐PDO/mol‐glycerol) from CG‐based rich/mineral media. Furthermore, fed‐batch fermentation with 50–80 g L−1 initial CG‐concentration was tested to enhance the PDO production. Among these, 60 g L−1 initial CG‐concentration assisted strain BOH3 in utilizing a high level of CG (~174 g L−1); it supported a higher‐titer of PDO production (103.55–116.45 g L−1), with the highest yield (0.72–0.81 mol−PDO/mol−glycerol) ever reported. CONCLUSION: Clostridium strain BOH3 shows the potential to support a high titer (>116 g L−1) with the highest yield (>0.80 mol mol−1) of PDO production from CG (a waste). It can be considered for industrial‐scale PDO production. © 2024 Society of Chemical Industry (SCI). [ABSTRACT FROM AUTHOR]

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

L-cysteine is a proteogenic amino acid with many applications in the pharmaceutical, food, animal feed, and cosmetic industries. Due to safety and environmental issues in extracting L-cysteine from animal hair and feathers, the fermentative production of L-cysteine offers an attractive alternative using renewable feedstocks. Strategies to improve microbial production hosts like Pantoea ananatis, Corynebacterium glutamicum, Pseudomonas sp., and Escherichia coli are summarized. Concerning the metabolic engineering strategies, the overexpression of feedback inhibition-insensitive L-serine O-acetyltransferase and weakening the degradation of L-cysteine through the removal of L-cysteine desulfhydrases are crucial adjustments. The overexpression of L-cysteine exporters is vital to overcome the toxicity caused by intracellular accumulating L-cysteine. In addition, we compiled the process engineering aspects for the bioproduction of L-cysteine. Utilizing the energy-efficient sulfur assimilation pathway via thiosulfate, fermenting cheap carbon sources, designing scalable, fed-batch processes with individual feedings of carbon and sulfur sources, and implementing efficient purification techniques are essential for the fermentative production of L-cysteine on an industrial scale. [ABSTRACT FROM AUTHOR]

Monoclonal antibodies (mAbs) used in biomedical research and therapeutic applications are primarily produced by Chinese hamster ovary (CHO) cells via fed-batch culture. The growing need for elevated quantities of biologics mandates the continual optimization of the mAb production process. The development of an effective process control method is indispensable for the production of specified mAbs by CHO cells. In this study, the effects of the pH control strategy on CHO cell fed-batch culture to produce an antibody (EA5) were first investigated in a 3 L bioreactor. The results indicate that controlling the culture pH at 7.2 during the fed-batch stage could produce a higher EA5 titer of 6.1 g/L with a lower Man5 ratio of 2.2% by day 14. Based on this, an optimized CHO cell fed-batch culture was conducted in a 15 L bioreactor to verify its effectiveness and stability. In this case, on day 14, an EA5 titer of 6.5 g/L was achieved with productivity of 0.46 g/L/day, which was 1.07-fold higher compared to that of the culture in the 3 L bioreactor. Furthermore, regarding the product quality, a monomer abundance of 96.0%, a main peak of 55.0%, and a Man5 proportion of 2.4% were maintained in the 15 L bioreactor. In addition, different cell clarification processes were evaluated using the CHO cell culture broth from the 3 L and 15 L bioreactors to further improve productivity and economic performance. Overall, this study provides some directions for process intensification and improving the quality of mAbs produced by CHO cells in the biopharmaceutical industry.

Process intensification has been widely used for many years in the mammalian biomanufacturing industry to increase productivity, agility and flexibility while reducing production costs. The most commonly used intensified processes are operated using a perfusion or fed‐batch seed bioreactor enabling a higher than usual seeding density in the fed‐batch production bioreactor. Hence, as part of the growth phase is shifted to the seed bioreactor, there is a lower split ratio, which increases the criticality of the seed bioreactor and could impact production performance. Therefore, such intensified processes should be designed and characterized for robust process scale‐up. This research work is focused on intensified processes with high seeding density inoculated from seed bioreactor in fed‐batch mode. The impact of the feeding strategy and specific power input (P/V) in the seed bioreactor and on the production step with two different cell lines (CL1 and CL2) producing two different monoclonal antibodies was investigated. Cell culture performance in the production bioreactor has been improved due to more stressful conditions for the cells in the seed bioreactor and the impact of the production bioreactor P/V on the production performance was limited. This is the first reported study highlighting a positive impact of cellular stress in seed bioreactors on intensified production bioreactor with the introduction of the "organized stress" concept. [ABSTRACT FROM AUTHOR]

L-cysteine is a proteogenic amino acid with many applications in the pharmaceutical, food, animal feed, and cosmetic industries. Due to safety and environmental issues in extracting L-cysteine from animal hair and feathers, the fermentative production of L-cysteine offers an attractive alternative using renewable feedstocks. Strategies to improve microbial production hosts like Pantoea ananatis, Corynebacterium glutamicum, Pseudomonas sp., and Escherichia coli are summarized. Concerning the metabolic engineering strategies, the overexpression of feedback inhibition-insensitive L-serine O-acetyltransferase and weakening the degradation of L-cysteine through the removal of L-cysteine desulfhydrases are crucial adjustments. The overexpression of L-cysteine exporters is vital to overcome the toxicity caused by intracellular accumulating L-cysteine. In addition, we compiled the process engineering aspects for the bioproduction of L-cysteine. Utilizing the energy-efficient sulfur assimilation pathway via thiosulfate, fermenting cheap carbon sources, designing scalable, fed-batch processes with individual feedings of carbon and sulfur sources, and implementing efficient purification techniques are essential for the fermentative production of L-cysteine on an industrial scale.

Non-toxic derivatives of the cholera toxin are extensively used in neuroscience, as neuronal tracers to reveal the location of cells in the central nervous system. They are, also, being developed as vaccine components and drug-delivery vehicles. Production of cholera-toxin derivatives is often non-reproducible; the quality and quantity require extensive fine-tuning to produce them in lab-scale settings. In our studies, we seek a resolution to this problem, by expanding the molecular toolbox of the Escherichia coli expression system with suitable production, purification, and offline analytics, to critically assess the quality of a probe or drug delivery, based on a non-toxic derivative of the cholera toxin. We present a re-engineered Cholera Toxin Complex (rCTC), wherein its toxic A1 domain was replaced with Maltose Binding Protein (MBP), as a model for an rCTC-based targeted-delivery vehicle. Here, we were able to improve the rCTC production by 11-fold (168 mg/L vs. 15 mg/L), in comparison to a host/vector combination that has been previously used (BL21(DE3) pTRBAB5-G1S). This 11-fold increase in the rCTC production capability was achieved by (1) substantial vector backbone modifications, (2) using Escherichia coli strains capable of growth-decoupling (V strains), (3) implementing a well-tuned fed-batch production protocol at a 1 L scale, and (4) testing the stability of the purified product. By an in-depth characterization of the production process, we revealed that secretion of rCTC across the E. coli Outer Membrane (OM) is processed by the Type II secretion-system general secretory pathway (gsp-operon) and that cholera toxin B-pentamerization is, likely, the rate-limiting step in complex formation. Upon successful manufacturing, we have validated the biological activity of rCTC, by measuring its binding affinity to its carbohydrate receptor GM1 oligosaccharide (Kd = 40 nM), or binding to Jurkat cells (93 pM) and delivering the cargo (MBP) in a retrograde fashion to the cell. [ABSTRACT FROM AUTHOR]

In this study, 7,10-dihydroxy-8(E)-octadecenoic acid (DOD) was produced using extracellular enzymes present in the supernatant of Pseudomonas aeruginosa. DOD concentration and productivity were improved using extracellular enzymes obtained from P. aeruginosa culture broth than using conventional whole cell culture method. Among the different carbon sources used during cell pre-culture, glycerol showed higher DOD production than using glucose or crude glycerol. In batch process, the highest concentration of DOD (8.82 g/L) was accomplished using supernatant derived from 12 h-grown pre-culture of P. aeruginosa strain after 72 h of bioconversion process. To further improve DOD production, a fed-batch process was used with the addition of surfactant and enzyme concentrate. The fed-batch process using a 4-fold concentrated enzyme solution with Tween 80 yielded the highest DOD concentration (27.5 g/L at 72 h) with an 8.28-fold increase in DOD production compared to using whole cells. The prominence of this study was that the highest DOD production (27.5 g/L) ever reported was achieved using extracellular enzymes via a fed-batch process. Another significant aspect of this study is the possibility of utilizing abundantly available crude glycerol substrate for DOD production, which will help direct the development of sustainable process. Therefore, the overall study is projected to speed up the development of DOD production using inexpensive substrates such as crude glycerol on a larger scale. [ABSTRACT FROM AUTHOR]

Chinese hamster ovary (CHO) cells are by far the most important mammalian cell lines used for producing antibodies and other therapeutic proteins. It is critical to fully understand their physiological conditions during a bioprocess in order to achieve the highest productivity and the desired product quality. Flow cytometry technology possesses unique advantages for measuring multiple cellular attributes for a given cell and examining changes in cell culture heterogeneity over time that can be used as metrics for enhanced process understanding and control strategy. Flow cytometry‐based assays were utilized to examine the progression of cell cycle and apoptosis in three case studies using different antibody‐producing CHO cell lines in both fed‐batch and perfusion bioprocesses. In our case studies, we found that G0/G1 phase distribution and early apoptosis accumulation responded to subtle changes in culture conditions, such as pH shifting or momentary glucose depletion. In a perfusion process, flow cytometry provided an insightful understanding of the cell physiological status under a hypothermic condition. More importantly, these changes in cell cycle and apoptosis were not detected by a routine trypan blue exclusion‐based cell counting and viability measurement. In summary, integration of flow cytometry into bioprocesses as a process analytical technology tool can be beneficial for establishing optimum process conditions and process control. [ABSTRACT FROM AUTHOR]

Abstract The cap protein is encoded by the orf2 gene of porcine circovirus type 2 (PCV2) has the main antigen epitope of PCV2 and can form virus-like particles (VLPs), which are expressed in insect cells. PCV2-VLPs can effectively inhibit PCV2 replication as a subunit vaccine. In this study, a robust and reliable fed-batch process was successfully developed for the production of PCV2-VLPs by Sf9 cells. The feeding solution, feeding strategy, and cell density at infection were optimized to maximize the final PCV2-VLPs production yields. The cell density at infection and the volumetric PCV2-VLPs production reached 12 × 106 cells/mL and 110 mg/L, respectively, which yielded 3- and 3.6-fold enhancements compared to the batch culture. The PCV2-VLPs produced in fed-batch culture were not different from the PCV2-VLPs produced in a batch culture in an immunity test. A highly efficient production process was produced for PCV2-VLPs subunit vaccines, which could provide an effective means for the industrial production of PCV2 vaccines.

This work presents theoretical evidence that the vacuferm technology can confer several advantages even when the fermentor is operated in fed-batch mode with small cycle times aiming to avoid the accumulation of potentially inhibitory side products. More specifically, a mathematical model for a fed-batch fermentor coupled with a vacuum flash tank for in situ ethanol recovery is developed and the simplest possible operational strategy is optimized within the context of maximizing the volumetric productivity of the process. Using numerical values obtained from the literature, it is demonstrated that significant increase in the volumetric productivity of ethanol (up to 33.5 kg EtOH m−3 h−1) can be achieved. Evidently, the results obtained indicate the potential to employ the proposed methodology to operate existing industrial facilities with minor modifications and equipment additions. [ABSTRACT FROM AUTHOR]

Botulinum neurotoxin has remarkably transitioned from a food safety hazard and biological warfare to an effective therapeutic drug. Currently, botulinum neurotoxins have seven serotypes (BoNT/A-G) in the form of protein complexes produced by Clostridium , a gram-positive and sporeforming bacteria. The conversion of toxins into useful drug substances of choice using the biotechnological process is tremendously increasing. Recent studies have shown that Botulinum neurotoxin-A (BoNT/A) has different biological activities and potencies in experimental and clinical conditions. They also indicate that the manufacturing process influences the potency and efficacy of BoNT/A drugs. Thus, this review focuses on the following criteria: detailed Fed-batch operation that includes the upstream and downstream processing of BoNT/A, the underlying mechanism behind the neurotoxic effect, and commercially available FDA-approved BoNT/A products and their therapeutic uses. Still, some research gaps exist in the mechanism for the treatment of psychiatric disorders., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

The molecular weight of hyaluronic acid (HA) is a critical property which determines its usage in various biomedical applications. This study investigates the correlation between the availability of a critical cofactor, acetyl-CoA, the concentration of a limiting precursor, UDP-N-acetylglucosamine (UDP-GlcNAc), and the molecular weight of HA (MWHA) produced by recombinant Lactococcus lactis MKG6 cultures. This strain expressed three heterologous HA-pathway genes obtained from the has operon of Streptococcus zooepidemicus in an ldh-mutant host strain, L. lactis NZ9020. A flux balance analysis, performed using the L. lactis genome-scale metabolic network, showed a positive correlation of acetyl-CoA flux with the UDP-GlcNAc flux and the experimental data on HA productivity. To increase the intracellular levels of acetyl-CoA, acetate was supplemented as a pulse feed in anaerobic batch cultures. However, acetate is effectively utilized only in the presence of glucose and exhaustion of glucose resulted in decreasing the final MWHA (1.5 MDa). Co-supplementation of acetate resulted in enhancing the acetyl-CoA and UDP-GlcNAc levels as well as the MWHA to 2.5 MDa. This logic was extended to fed-batch cultures, designed with a pH-based feedback control of glucose feeding and pulse acetate supplementation. When the glucose feed concentration was optimally adjusted to prevent glucose exhaustion or accumulation, the acetate utilization was found to be high, resulting in significantly enhanced levels of acetyl-CoA and UDP-GlcNAc as well as a MWHA of 3.4 MDa, which was sustained at this value throughout the process. This study provides the possibility of commercially producing high MWHA using recombinant L. lactis strains. [ABSTRACT FROM AUTHOR]

Microalgae are unicellular and photosynthetic organisms which have great potential for providing various products of interest in the food, chemicals, pharmaceuticals, and cosmetics industry. The green alga Ankistrodesmus braunii has been cited as capable of producing large amounts of lipids. In this sense, this work aims to study A. braunii growth in tubular photobioreactor using different amounts of nitrogen and under different cultivation processes. In batch cultures, the maximum biomass concentration (Xm) was 1588 ± 11 mg L−1 using 20 mM of NaNO3. A fed-batch process with the addition of 20 mM NaNO3 each 48 h from the first to the sixth cultivation day reached Xm = 2753 ± 7 mg L−1. The semi-continuous process was effective to eliminate the lag phase, allowing to obtain Xm = 2399 ± 5 mg L−1. In this condition, the protein and lipid levels in the biomass were 33.1 ± 0.2% and 38.6 ± 0.2%, respectively. The maximum specific growth rate (μm) reached a maximum value of 0.998 day−1 in the semi-continuous process using 20 mM NaNO3. The results of this study show the potential for the production in a large scale of A. braunii as a source of protein and lipids for commercial applications. [ABSTRACT FROM AUTHOR]

Highlights • The Performance of two suspension and serum-free adapted cell lines was evaluated for the production of recombinant Vesicular stomatitis virus. • There was a positive correlation between volumetric virus titer and cell density up to 2E+07 cells per ml. • At small scale 293SF-3F6 cells produced VSV-GFP at a volumetric titer of 8.1E+10 extracellular infectious particles/ml. • At 200 ml scale, 293SF-3F6 cells produced VSV-GFP at a volumetric titer of 2.9E+10 extracellular infectious particles/ml. Abstract During the last decade, oncolytic viruses such as vesicular stomatitis virus (VSV) have gained tremendous popularity as efficient vaccines for infectious diseases as well as for the treatment of cancer. Our laboratory has developed two stable cell lines, 293SF-3F6 (derived from HEK293A cells) and SF-BMAdR cells (a variant of A549 that expresses the E1 region of human adenovirus). These two cell lines were adapted to grow efficiently in suspension culture and in serum-free medium. In this report we evaluated the production of a recombinant VSV expressing the green fluorescent protein (VSV-GFP) in these two stable cell lines. At a relatively low cell density of 500,000 cells per ml, 293SF-3F6 produced 4.6 times more infectious particles than SF-BMAdR cells. There was a positive correlation between volumetric virus titer and cell density up to 2.E + 07 cells/ml. A fed-batch process using an in-house medium and feed was developed to support the growth of 293SF-3F6 cells up to a concentration of 1.E + 07 cells/ml for infection at higher cell density and VSV production at high titer. Shifting the temperature from 37 °C to 34 °C at infection time improved VSV titer up to 3.3 fold. After scaling up the optimal condition from small scale (3 ml) to larger volumes (50 & 200 ml), the maximal volumetric titer obtained using the 293SF-3F6 cells was in average 2.9E + 10 extracellular infectious particles/ml. In conclusion, our data demonstrated that 293SF-3F6 cells, for which a cGMP master cell bank is available, is a performant cell line to scale up VSV production in suspension culture using serum-free medium. [ABSTRACT FROM AUTHOR]

Abstract A fed-batch approach in stirred tank bioreactors for achieving enzymatic hydrolysis with a high load of lignocellulosic mass is described in this paper. This approach allows ease of mixing, increasing the load of a higher concentration of biomass without performance decrease, in comparison with the typical batch process, and consequently it provides a higher concentration of reducing sugars for a down-stream fermentation process; additionally, the fed-batch process times are shorter than the ones that have been reported so far. The work basically consisted of experiments in fed-batch operation with final sorghum straw loadings of 5, 10, 15 and 20% w v−1, using a commercial enzyme complex, and conventional batch experiments for comparison purposes. Without typical operation problems of batch processes such as the lack of effective initial mixing and difficult adjustment of pH and temperature, hydrolysate broths up to a reducing sugars concentration of 130 g L−1 were obtained. Finally, hydrolysates were fermented to verify effective bioethanol production, reaching concentrations of bioethanol of 48 g L−1. Graphical abstract Image 1 Highlights • Fed-batch approach reduces operational problems caused by poor mixing. • Fed-batch approach enables fast adjustment of pH and temperature. • Fed-batch experiments of 5, 10, and 15% w v−1 reach a holocellulose yield over 90%. • Fed-batch experiment of 20% w v−1 reaches a holocellulose yield of 75%. • Fermentations from hydrolysates reach ethanol concentrations higher than 40 g L−1. [ABSTRACT FROM AUTHOR]

Highlights • Fed-batch strategy allowed to increase solid loadings up to 30% (w/v) in enzymatic hydrolysis. • Higher glucose concentrations were reached in fed-batch enzymatic hydrolysis operation. • Less powerful motor is required in fed-batch enzymatic hydrolysis. • Fed-batch operational mode presented better performance in cellulose hydrolysis. • Rheological behavior and power consumption were optimized at 30% (w/v) of solid loadings. Abstract This work describes improvements in the enzymatic hydrolysis of hydrothermally pretreated sugarcane straw. The experiments were performed in batch and fed-batch operation modes (50 mL and 3 L working volumes) with final solid loadings of 10, 20, and 30% (w/v) and enzyme dosage of 10 FPU/g substrate. The fed-batch assays showed the most promising results in terms of glucose production and cellulose-to-glucose conversion. In the experiments using a 3 L working volume, analysis of cellulose-to-glucose conversion, power consumption, and apparent viscosity showed that the best assays were those with smoother feed delivery (substrate plus enzyme) throughout the enzymatic hydrolysis. This strategy resulted in 108.8 g/L of glucose and cellulose-to-glucose conversion of 59.7%, corresponding to a final theoretical ethanol content of 55.6 g/L. The power consumption per unit volume and the apparent viscosity were less than 5 kW/m3 and 35 mPa s, respectively, throughout the hydrolysis. Thus, a more concentrated liquor and a less powerful motor to deliver power during the hydrolysis process were obtained in fed-batch strategy reducing the process energy costs. [ABSTRACT FROM AUTHOR]

To make the bioethanol conversion process economically viable, improving solids loading (>15% [w/v]) in different unit operations is crucial. In this study, alkali pretreatment of sugarcane bagasse was carried out at high solids loading. Temperature and pH were optimised via batch simultaneous saccharification and fermentation (SSF) experiments using Saccharomyces cerevisiae Y‐2034 or Kluyveromyces marxianus NCYC‐587. The effects of enzyme addition mode and solids loading on ethanol production were studied in fed‐batch SSF experiments. It was demonstrated that the optimised pH and temperature for both S. cerevisiae Y‐2034 and K. marxianus NCYC‐587 were 5.2 and 37°C. The mode of enzyme addition "one‐time dose" improved ethanol productivity better than the "batch addition" mode. With fresh substrates added, solids loading strongly increased from 19% to 33% (w/v), corresponded to a small decline in the ethanol yield. At the final solids loading of 33% (w/v), the highest ethanol concentration of 75.57 ± 2.14 g/L was obtained, with ethanol productivity of 0.63 ± 0.02 g/(L h) and a yield of 66.17 ± 1.87%. Sugarcane bagasse was pretreated with alkali solutions at high solids loading. Fed‐batch simultaneous saccharification and fermentation experiments were conducted at three high solids loadings. The highest ethanol concentration of 75.57 g/L was obtained. [ABSTRACT FROM AUTHOR]

l‐tryptophan is an essential amino acid of high industrial interest that is routinely produced by microbial processes from glucose as carbon source. Glycerol is an alternative substrate providing a variety of economic and metabolic advantages. Process performance of the recombinant l‐tryptophan producer Escherichia coli NT367 was studied in controlled fed‐batch processes. The chromosome of the recombinant l‐tryptophan producer was equipped with additional genes coding for enzymes of the aromatic amino acids biosynthetic pathway and l‐serine biosynthesis, including genes for feedback‐resistant enzyme variants (trpEfbr, aroFBL, and serAfbr), deletions of enzymatic steps for the degradation of precursors or the product l‐tryptophan (sdaB and tnaA), and alterations in the regulation of l‐tryptophan metabolism (deletion of trpL and trpR). The impact of glycerol supply rates as well as the application of a multicopy plasmid (pF112‐ aroFBL ‐kan) were investigated in fully controlled stirred‐tank bioreactors on a 15 L scale. The combination of E. coli NT367 carrying pF112‐ aroFBL ‐kan and an appropriate biomass‐specific glycerol supply‐rate resulted in the highest final product concentration of 12.5 g L −1l‐tryptophan with the lowest concentrations of other aromatic amino acids. Fed‐batch production of l‐tryptophan from glycerol was shown for the first time with recombinant E. coli. L‐tryptophan is an essential amino acid of high industrial interest that is routinely produced by microbial processes from glucose as carbon source. Glycerol is an alternative substrate providing a variety of economic and metabolic advantages. Process performance of variants of the recombinant L‐tryptophan producer Escherichia coli NT367 was studied in controlled fed‐batch processes. [ABSTRACT FROM AUTHOR]

Alkali-pretreated sugarcane bagasse fiber was subjected to fed-batch simultaneous saccharification and fermentation (SSF) with a pre-hydrolysis process to increase the solids loading and produce a high concentration of ethanol. The hydrolysis medium and yeast feeding modes were investigated to determine suitable conditions for high sugar yield and ethanol production. Batch addition resulted in a cumulative substrate concentration of up to 36% (w/v) and enhanced ethanol concentrations, while ethanol conversion efficiency gradually declined. Enzymatic pre-hydrolysis and fermentation with fed-batch mode contributed to the SSF process. The highest ethanol concentration was 66.915 g/L with the conversion efficiency of 72.89%, which was achieved at 30% (w/v) solids content after 96 h of fermentation. Hydrolyzed medium and yeast were added in batch mode at 24 h of enzymatic hydrolysis and fermentation, respectively. Thus, combining the fed-batch mode with pre-hydrolysis SSF produced a high yield of ethanol.

The present study compared dilute acid and alkaline pretreatment of corn husk. It showed that maximum reducing sugar production was obtained when biomass was pretreated using dilute sodium hydroxide. Further, effectiveness of dilute alkaline pretreatment was evaluated through Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), scanning electron microscopy (SEM), and biochemical composition study. Batch enzymatic hydrolysis of dilute alkaline pretreated biomass was carried out using several combinations of different cellulases (Trichoderma, Fusarium, and Aspergillus). Maximum reducing sugar production was obtained when pretreated biomass was hydrolyzed using mixture of Trichoderma, Fusarium, and Aspergillus cellulases. Further, batch enzymatic hydrolysis process was optimized using CCD (central composite design)-based RSM (response surface methodology). In addition, using mixture of different cellulases, batch and fed-batch enzymatic hydrolysis processes were compared. It showed that 14.34-mg/mL higher reducing sugar production was obtained in the case of fed-batch process compared to batch process. [ABSTRACT FROM AUTHOR]

In this paper, we propose a nonlinear switched control system for microbial 1,3-propanediol (1,3-PD) fed-batch production, where the smooth feeding rate of glycerol as well as the switching instants between batch process and feeding process are taken as control variables. To maximize 1,3-PD yield and minimize the fluctuation of feeding rate, we then present an optimal switching control problem with a cost on the smooth feeding rate variation and subject to path constraints reflecting the operational requirements. For solving this problem, we convert it to a series of parameter optimization problems by using a novel control parameterization scheme, a time-scaling transformation and a constraint transcription technique. An analytical expression of the total variation for the smooth feeding rate is also derived. On this basis, we develop a parallel Particle Swarm Optimization (PSO) algorithm together with the gradient-based optimization to solve the resulting problem. Finally, numerical simulations indicate that sacrificing a small amount of 1,3-PD can reduce the feeding rate variation significantly. [ABSTRACT FROM AUTHOR]

ABSTRACT Coagulation factor II (prothrombin; FII) is the pre-proteolyzed precursor to thrombin in the coagulation cascade. It has 10 sites of gamma-carboxylation, which are required for its bioactivity, and is N-glycosylated at three of four putative sites. Production of recombinant human FII (rhFII) using a platform fed-batch process designed for monoclonal antibody production resulted in low levels of gamma-carboxylation and sialylation. There have not been any prior reports of successful process development and clinical manufacture of rhFII with optimal, consistent gamma-carboxylation and sialylation. In order to develop such a fed-batch process, various process parameters were evaluated to determine their impact on product quality. Process temperature and temperature shift timing were important for both sialic acid level and gamma-carboxyglutamate (Gla) level. In addition, vitamin K concentration and the type of surfactant used for preparation of vitamin K stock solution were also important for gamma carboxylation. A fed-batch study performed with various medium additives known to be involved in the N-glycosylation pathway, such as N-acetyl- d-mannosamine (ManNAc), galactose (Gal), dexamethasone, and manganese sulfate, increased the level of sialylation and enabled the elucidation of some potential bottlenecks in the sialylation pathway. The optimized process based on these studies yielded a reduction in the level of missing Gla by 0.4 moles per mole of rhFII in cell culture and a nearly threefold increase in sialic acid level. The process was successfully implemented at the 2000 L scale where a high Gla level and sialylation levels were achieved in all GMP lots. Biotechnol. Bioeng. 2017;114: 1991-2000. © 2017 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

This paper considers optimal control of glycerol producing 1,3-propanediol (1,3-PD) via microbial fed-batch fermentation. The fed-batch process is formulated as a nonlinear switched time-delay system. In general, the time-delay in the fed-batch process cannot be exactly estimated. Our goal is to design an optimal switching control scheme to simultaneously maximize 1,3-PD productivity and 1,3-PD yield under time-delay uncertainty. Accordingly, we propose a robust multi-objective optimal switching control model, in which two objectives, i.e., 1,3-PD productivity and 1,3-PD yield, and their sensitivities with respect to uncertain time-delay are considered in the objective vector. The control variables in this problem are the feeding rate of glycerol, the switching instants and the terminal time of the process. By introducing an auxiliary dynamic system to calculate the objective sensitivities and performing a time-scaling transformation, we obtain an equivalent multi-objective optimal switching control problem in standard form. We then convert the equivalent multi-objective optimal control problem into a sequence of single-objective optimal switching control problems by using a modified normal boundary intersection method. A novel gradient-based single-objective solver combining control parameterization with constraint transcription technique is developed to solve these resulting single-objective optimal control problems. Finally, numerical results are provided to verify the effectiveness of the proposed solution approach. [ABSTRACT FROM AUTHOR]

Enrofloxacin is a synthetic second-generation fluoroquinolone used as an antimicrobial agent exclusively in veterinary medicine. To simulate the treatment of wastewater contaminated by enrofloxacin, four-day long fed-batch runs were carried out according to the Fenton process with an enrofloxacin solution as model, to which hydrogen peroxide and ferrous ion were added twice a day. The residual enrofloxacin concentration was practically coincident to that detected at the end of the batch tests. Hydrogen peroxide was almost completely consumed after each feeding period, while the total organic carbon (TOC) concentration decreased gradually within three days, corresponding to a reduction > 58 %. From the third day on, the TOC falling rate was quite low. A yellow sludge settled due to the precipitation of both Fe(OH)3 and a complex formed by ferric ion with adsorbed enrofloxacin and/or its oxidation products. [ABSTRACT FROM AUTHOR]

ABSTRACT We present an integrated framework for the online optimal experimental re-design applied to parallel nonlinear dynamic processes that aims to precisely estimate the parameter set of macro kinetic growth models with minimal experimental effort. This provides a systematic solution for rapid validation of a specific model to new strains, mutants, or products. In biosciences, this is especially important as model identification is a long and laborious process which is continuing to limit the use of mathematical modeling in this field. The strength of this approach is demonstrated by fitting a macro-kinetic differential equation model for Escherichia coli fed-batch processes after 6 h of cultivation. The system includes two fully-automated liquid handling robots; one containing eight mini-bioreactors and another used for automated at-line analyses, which allows for the immediate use of the available data in the modeling environment. As a result, the experiment can be continually re-designed while the cultivations are running using the information generated by periodical parameter estimations. The advantages of an online re-computation of the optimal experiment are proven by a 50-fold lower average coefficient of variation on the parameter estimates compared to the sequential method (4.83% instead of 235.86%). The success obtained in such a complex system is a further step towards a more efficient computer aided bioprocess development. Biotechnol. Bioeng. 2017;114: 610-619. © 2016 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Maximization of productivity and minimization of consumption are two top priorities for biotechnological industry. In this paper, we model a fed-batch process as a nonlinear switched time-delay system. Taking the productivity of target product and the consumption rate of substrate as the objective functions, we present a multi-objective optimization problem involving the nonlinear switched time-delay system and subject to continuous state inequality constraints. To solve the multi-objective optimization problem, we first convert the problem into a sequence of single-objective optimization problems by using convex weighted sum and normal boundary intersection methods. A gradient-based single-objective solver incorporating constraint transcription technique is then developed to solve these single-objective optimization problems. Finally, a numerical example is provided to verify the effectiveness of the numerical solution approach. Numerical results show that the normal boundary intersection method in conjunction with the developed single-objective solver is more favourable than the convex weighted sum method. [ABSTRACT FROM AUTHOR]

Pure and crude glycerol from biodiesel production have been used as substrates for citric acid production by acetate-negative mutants of Yarrowia lipolytica in fed-batch fermentation. Both the final concentration and the yield of the product were the highest when Y. lipolytica Wratislavia AWG7 strain was used in the culture with pure or crude glycerol. With a medium containing 200 g/L of glycerol, production reached a maximum of citric acid of 139 g/L after 120 h. This high yield of the product (up to 0.69 g of citric acid per gram of glycerol consumed) was achieved with both pure and crude glycerol. Lower yield of citric acid in the culture with Y. lipolytica Wratislavia K1 strain (about 0.45 g/g) resulted from increased erythritol concentrations (up to 40 g/L), accumulated simultaneously with the citric acid. The concentration of isocitric acid, a by-product in this fermentation, was very low, in the range from 2.6 to 4.6 g/L.

Free software for entering and documenting data EpiData is here used for design of a system for functional states recognition during a fermentation process. The identification of the current process state is based on the predetermined rules, rendering specific metabolic mechanisms. Developed system is further applied for a fed-batch cultivation of Saccharomyces cerevisiae.

Free software for entering and documenting data EpiData is here used for design of an advisory system for on-line control of a fermentation process. Based on the preliminary developed system for functional state recognition, presented here system will advise the user which new functional state can be reached and what kind of control actions have to be taken. New-designed system appears as an expert system and comprises knowledge of well-trained operators of cultivation processes. Developed advisory system is further applied for a fed-batch cultivation of Saccharomyces cerevisiae.

This paper deals with the design of feeding rates for dual-substrate fed-batch processes where the main control objective is the regulation of the microbial growth rate. To this end, feedback of the growth rate error is incorporated to a biomass proportional dual feeding law. A second-order sliding mode observer is used to estimate the growth rate, so that no additional sensors are required. Stability conditions are derived and robustness against several disturbances such as yield uncertainty, measurement errors and kinetic model mismatch is analytically and numerically evaluated. The advantages of the proposal include: minimal measurement requirements, regulation with fast convergence to the desired growth rate and reduced regulation error in the presence of disturbances. [ABSTRACT FROM AUTHOR]

The minimization of costs in the distillation step of lignocellulosic ethanol production requires the use of a high solids loading during the enzymatic hydrolysis to obtain a more concentrated glucose liquor. However, this increase in biomass can lead to problems including increased mass and heat transfer resistance, decreased cellulose conversion, and increased apparent viscosity with the associated increase in power consumption. The use of fed-batch operation offers a promising way to circumvent these problems. In this study, one batch and four fed-batch strategies for solids and/or enzyme feeding during the enzymatic hydrolysis of sugarcane bagasse were evaluated. Determinations of glucose concentration, power consumption, and apparent viscosity were made throughout the experiments, and the different strategies were compared in terms of energy efficiency (mass of glucose produced according to the energy consumed). The best energy efficiency was obtained for the strategy in which substrate and enzyme were added simultaneously (0.35 kg kWh). This value was 52 % higher than obtained in batch operation. [ABSTRACT FROM AUTHOR]

Hydrogen produced by microorganisms has been considered as a potential solution for sustainable hydrogen production for the future. In the current study, an advanced real-time optimisation methodology is developed to maximise the productivity of a 21-day fed-batch cyanobacterial hydrogen production process, which to the best of our knowledge has not been addressed before. This methodology consists of an economic model predictive control formulation used to predict the future experimental performance and identify the future optimal control actions, and a finite-data window least-squares procedure to re-estimate model parameter values of the on-going process and ensure the high accuracy of the dynamic model. To explore the efficiency of the current optimisation methodology, effects of its essential factors including control position, prediction horizon length, estimation window length, model synchronising frequency, terminal region and terminal cost on hydrogen production have been analysed. Finally, by implementing the proposed optimisation strategy into the current computational fed-batch experiment, a significant increase of 28.7% on hydrogen productivity is achieved compared to the previous study. [ABSTRACT FROM AUTHOR]

The effect of feeding methods on the production of astaxanthin by the yeast Phaffia rhodozyma ATCC 24202 was studied, using continuous and pulsed fed-batch processes and low cost materials as substrates (sugar cane juice and urea). In continuous fed-batch processes, a cellular astaxanthin concentration of 383.73 µg/g biomass was obtained. But in pulsed fed-batch processes a reduction in the cellular astaxanthin concentration (303.34 µg/g biomass) was observed. Thus the continuous fed-batch processes could be an alternative to industrial production of astaxanthin, allowing an increase in the biomass productivity without losses on astaxanthin production by the yeast.O efeito da alimentação na produção de astaxantina pela levedura Phaffia rhodozyma ATCC 24202 foi estudado, utilizando processos descontínuo alimentado com alimentação contínua e intermitente, e matérias-primas de baixo custo como substratos (caldo de cana de açúcar e uréia). Em processos descontínuo alimentado com alimentação contínua, uma concentração celular de astaxantina de 383,73 µg/g biomassa foi obtida. Entretanto, em processos descontínuo alimentado com alimentação intermitente, uma redução na concentração celular de astaxantina (303,34 µg/g biomassa) foi observada. Desta forma, processos descontínuo alimentado com alimentação contínua poderiam ser uma alternativa na produção industrial de astaxantina, permitindo um aumento na produtividade de biomassa sem perdas na produção de astaxantina pela levedura.

Arthrospira platensis has been photoautotrophically cultivated for the production of high quality biomass. It contains satisfactory contents of protein, pigments, and fatty acids. The use of organic compounds as a carbon source has been studied, aiming to improve the biomass productivity in a mixotrophic process. In this study, A. platensis was cultivated by fed-batch process with the addition of sodium acetate, evaluating the effect of different concentrations and feeding time of this organic carbon source by the use of response surface methodology. Addition of sodium acetate was shown to be suitable for increasing the maximum cell concentration. The optimum condition was estimated by the model to be achieved with 387 mg Ld of sodium acetate for 6.5 days. Employing this condition, average X of 1769 ± 71 mg L was achieved. This X value had an increase of almost 39 % in comparison with the standard cultivation, without acetate addition (1275 mg L). [ABSTRACT FROM AUTHOR]

Three strategies were presented to achieve high solids loading while maximizing carbohydrate conversion, which are fed-batch, splitting/thickening, and clarifier processes. Enzymatic hydrolysis was performed at water insoluble solids (WIS) of 15% using washed dilute-acid pretreated corn stover. The carbohydrate concentration increased from 31.8 to 99.3 g/L when the insoluble solids content increased from 5% to 15% WIS, while the final carbohydrate conversion was decreased from 78.4% to 73.2%. For the fed-batch process, a carbohydrate conversion efficiency of 76.8% was achieved when solid was split into 60:20:20 ratio, with all enzymes added first. For the splitting/thickening process, a carbohydrate conversion of 76.5% was realized when the filtrate was recycled to simulate a steady-state process. Lastly, the clarifier process was evaluated and the highest carbohydrate conversion of 81.4% was achieved. All of these results suggests the possibility of enzymatic hydrolysis at high solids to make the overall conversion cost-competitive. [ABSTRACT FROM AUTHOR]

In this paper, we propose a switched autonomous system with time delay to model the 1,3-propanediol (1,3-PD) production in a fed-batch process. Taking the switching instants and the terminal time as the control variables, we formulate a constrained time-delayed optimal control (CTOC) problem to optimize the 1,3-PD production process. Using a time-scaling transformation and parametrizing the switching instants into new parameters, an equivalent CTOC problem is investigated. A numerical solution method is then developed to seek the optimal control strategy. This method is based on the constraint transcription technique and the gradients of the cost functional together with those of the constraints. Numerical simulation results show that the mass of 1,3-PD per unit time at the terminal time is increased considerably compared with the experiment results. [ABSTRACT FROM AUTHOR]

Fed-batch enzymatic hydrolysis process from alkali-pretreated sugarcane bagasse was investigated to increase solids loading, produce high-concentration fermentable sugar and finally to reduce the cost of the production process. The optimal initial solids loading, feeding time and quantities were examined. The hydrolysis system was initiated with 12% (w/v) solids loading in flasks, where 7% fresh solids were fed consecutively at 6 h, 12 h, 24 h to get a final solids loading of 33%. All the requested cellulase loading (10 FPU/g substrate) was added completely at the beginning of hydrolysis reaction. After 120 h of hydrolysis, the maximal concentrations of cellobiose, glucose and xylose obtained were 9.376 g/L, 129.50 g/L, 56.03 g/L, respectively. The final total glucan conversion rate attained to 60% from this fed-batch process. [ABSTRACT FROM AUTHOR]

Frutalin, a galactose-specific lectin used to detect specific tumour markers, is a protein with low expression level in breadfruit. In the present study, fed-batch fermentation in a stirred tank bioreactor was used as a strategy to enhance protein production by a recombinant Pichia pastoris KM71H. By using this process, the production of recombinant frutalin was 4-fold higher than the value obtained in shaker flasks batch assays. Supplementation of the fermentation medium with trace elements ( Pichia trace minerals, PTM) was also evaluated in order to stimulate production of the recombinant protein. The addition of PTM to the minimum medium afforded a recombinant protein production of 13.4 mg L, which was 2.5-fold higher than that achieved from the culture medium without PTM supplementation. These results are significant as the development of strategies to improve the production of recombinant frutalin may broaden its application in cancer diagnosis. [ABSTRACT FROM AUTHOR]

The effects of varying initial concentrations of microcrystalline cellulose on cellulase production with Trichoderma reesei RUT-C30 as well as the effects of varying lactose and ammonium sulfate concentrations in the feed medium were studied simultaneously in parallel-operated shake flasks and, alternatively, in parallel-operated stirred-tank bioreactors on a 10-mL scale. Fifteen experiments were performed as triplicates in shake flasks as well as in stirred-tank bioreactors in parallel to identify the parameters of second-order polynomials for the estimation of the final filter paper activity of T. reesei RUT-C30 after a process time of 96 h. Even though parameter estimation was not possible based on the results of the shake flasks due to final enzyme activities at or below the detection limit (with the exception of one shake flask), the identification of the second-order polynomial was successful with the results of the parallel-operated stirred-tank bioreactors on a 10-mL scale. Reaction conditions with 53.3 g L microcrystalline cellulose in the initial medium, no lactose feeding and 3.3 g L day intermittent ammonium sulfate addition were estimated to be optimal. The final experimental validation of the optimum substrate supply on a L-scale resulted in the production of 4.88 filter paper units (FPU) mL with T. reesei RUT-C30 after 96 h. This is an improvement by a factor of 3.6 compared to the reference batch process (1.35 FPU mL). [ABSTRACT FROM AUTHOR]

A serum-free medium (CHO-SFM) together with a fed-batch process was developed for the cultivation of a recombinant GS-CHO cell line producing TNFR-Fc. According to the metabolic characteristics of GS-CHO cell, a basal medium was prepared by supplementing DMEM:F12:RPMI1640 (2:1:1) with amino acids, insulin, transferrin, Pluronic F68 and some other ingredients. Statistical optimization approaches based on Plackett-Burman and central composite designs were then adopted to identify additional positive determinants and determine their optimal concentrations, which resulted in the final CHO-SFM medium formulations. The maximum antibody titer reached was 90.95 mg/l in the developed CHO-SFM, which was a 18 % and 10 fold higher than that observed in the commercial EX-CELL™ 302 medium (76.95 mg/l) and basal medium (8.28 mg/l), respectively. Subsequently, a reliable, reproducible and robust fed-batch strategy was designed according to the offline measurement of glucose, giving a final antibody yield of 378 mg/l, which was a threefold improvement over that in conventional batch culture (122 mg/l) using CHO-SFM. In conclusion, the use of design of experiment (DoE) method facilitated the development of CHO-SFM medium and fed-batch process for the production of recombinant antibody using GS-CHO cells. [ABSTRACT FROM AUTHOR]

Abstract: Formiline pretreatment pertains to a biomass fractionation process. In the present work, Formiline-pretreated sugarcane bagasse was hydrolyzed with cellulases by batch and multi-step fed-batch processes at 20% solid loading. For wet pulp, after 144h incubation with cellulase loading of 10FPU/g dry solid, fed-batch process obtained ∼150g/L glucose and ∼80% glucan conversion, while batch process obtained ∼130g/L glucose with corresponding ∼70% glucan conversion. Solid loading could be further increased to 30% for the acetone-dried pulp. By fed-batch hydrolysis of the dried pulp in pH4.8 buffer solution, glucose concentration could be 247.3±1.6g/L with corresponding 86.1±0.6% glucan conversion. The enzymatic hydrolyzates could be well converted to ethanol by a subsequent fermentation using Saccharomices cerevisiae with ethanol titer of 60–70g/L. Batch and fed-batch SSF indicated that Formiline-pretreated substrate showed excellent fermentability. The final ethanol concentration was 80g/L with corresponding 82.7% of theoretical yield. [Copyright &y& Elsevier]