pH Control Strategy Research Articles

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

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

Process optimized for production of iturin A in biofilm reactor by Bacillus velezensis ND.

In this research, to provide an optimal growth medium for the production of iturin A, the concentrations of key amino acid precursors were optimized in shake flask cultures using the response surface method. The optimized medium were applied in a biofilm reactor for batch fermentation, resulting in enhanced production of iturin A. On this basis, a step-wise pH control strategy and a combined step-wise pH and temperature control strategy were introduced to further improve the production of iturin A. Finally, the fed-batch fermentation was performed based on combined step-wise pH and temperature control. The titer and productivity of iturin A reached 7.86 ± 0.23g/L and 65.50 ± 1.92mg/L/h, respectively, which were 37.65 and 65.20% higher than that before process optimization.

Optimisation of pH for minimising juice degradation during evaporation

During juice evaporation, sucrose loss can occur due to degradation reactions that are influenced by heat, pH, residence time, and dry substance content. The impacts of heat can be controlled through process and equipment design, and juice pH could be optimised to reduce its effects on degradation reactions. A factory sampling program was conducted with juices collected across an evaporator station comprised of six Robert (rising film) effects in series for a range of juice pH levels and analysed for sucrose loss and overall pH drop from clarified juice to syrup. Measured losses were compared to predictive correlations and showed strong agreement. Using the factory data, two pH control strategies to minimise sucrose degradation and the subsequent impacts on downstream processing were simulated with predictive correlations. The modelling results highlight the potential benefits that can be achieved.

Ammoniacal nitrogen recovery from swine slurry using a gas-permeable membrane: pH control strategies and feed-to-trapping volume ratio.

Gas-permeable membrane (GPM) technology is gaining interest to recover nitrogen from residual effluents due to its effectiveness, simple operation and capacity of producing a nutrient rich product with fertilising value. In this study, a GPM contactor was used at 25°C to recover total ammoniacal nitrogen (TAN) from swine slurry as a concentrated (NH4)2SO4 solution. Firstly, a synthetic solution was tested on a wide pH range (6-12). Results showed that the ammonia mass transfer constants (Km) increased from 7.9·10-9 to 1.2·10-6m/s as the pH increased. The reagent consumption to control the pH per mole nitrogen recovered had a minimum at pH 9, which showed a Km value of 3.0·10-7m/s. Secondly, various pH control strategies were tested using swine slurry, including (i) no pH control, (ii) pH control at 8.5, 9.0 and 10.0, and (iii) an initial spike of the NaOH equivalent to the required to control the pH at 9. The test without pH control reached a TAN recovery of around 60%, which could be an interesting strategy when high nitrogen recoveries or short operating times are not required. The pH control at 9 stood out as the most favourable operating condition due to its high Km and lower reagent consumption. Thirdly, several feed-to-trapping volume ratios ranging from 1:1 to 15:1 were tested using swine slurry at pH 9. These assays revealed that a GPM process with a high feed-to-trapping volume ratio fastens the recovery of 99% of TAN as a high purity (NH4)2SO4 solution containing 40g N/L.

Oxidative stress resistance prompts pyrroloquinoline quinone biosynthesis in Hyphomicrobium denitrificans H4-45

Pyrroloquinoline quinone (PQQ) is a natural antioxidant with diverse applications in food and pharmaceutical industries. A lot of effort has been devoted toward the discovery of PQQ high-producing microbial species and characterization of biosynthesis, but it is still challenging to achieve a high PQQ yield. In this study, a combined strategy of random mutagenesis and adaptive laboratory evolution (ALE) with fermentation optimization was applied to improve PQQ production in Hyphomicrobium denitrificans H4-45. A mutant strain AE-9 was obtained after nearly 400 generations of UV-LiCl mutagenesis, followed by an ALE process, which was conducted with a consecutive increase of oxidative stress generated by kanamycin, sodium sulfide, and potassium tellurite. In the flask culture condition, the PQQ production in mutant strain AE-9 had an 80.4% increase, and the cell density increased by 14.9% when compared with that of the initial strain H4-45. Moreover, batch and fed-batch fermentation processes were optimized to further improve PQQ production by pH control strategy, methanol and H2O2 feed flow, and segmented fermentation process. Finally, the highest PQQ production and productivity of the mutant strain AE-9 reached 307 mg/L and 4.26 mg/L/h in a 3.7-L bioreactor, respectively. Whole genome sequencing analysis showed that genetic mutations in the ftfL gene and thiC gene might contribute to improving PQQ production by enhancing methanol consumption and cell growth in the AE-9 strain. Our study provided a systematic strategy to obtain a PQQ high-producing mutant strain and achieve high production of PQQ in fermentation. These practical methods could be applicable to improve the production of other antioxidant compounds with uncleared regulation mechanisms.Key points• Improvement of PQQ production by UV-LiCl mutagenesis combined with adaptive laboratory evolution (ALE) and fermentation optimization.• A consecutive increase of oxidative stress could be used as the antagonistic factor for ALE to enhance PQQ production.• Mutations in the ftfL gene and thiC gene indicated that PQQ production might be increased by enhancing methanol consumption and cell growth.

Autotrophic nitrogen polishing of secondary effluents: Alkaline pH and residual nitrate control S0-driven denitratation for downstream anammox treatment

Energy-lean nitrogen removal technologies, such as partial nitritation/anammox, often encounter effluent issues due to elevated nitrate and ammonium levels. This study proposed a novel autotrophic polishing strategy coupling sulfur-driven denitratation with anammox. To explore the denitratation potential in obtaining stable and sufficient nitrite accumulation, the effects of pH, residual nitrate level, and biomass-specific nitrate loading rate (BSNLR) were investigated in an S0-packed bed reactor at low hydraulic retention time (i.e., 0.2 h). Implementing pH and residual nitrate control strategies would be easier in practice than BSNLR control to polish secondary effluent. Alkaline pH values could realize successful nitrite accumulation without residual nitrate, and further intensify the accumulation under increased residual nitrate levels. The nitrate level was positively correlated with the nitrite accumulation efficiency. At pH 8.5 and nitrate concentration of 1.0 ± 0.8 mg N L−1, sulfur-driven denitratation could successfully maintain nitrite accumulation of 6.4 ± 1.0 mg NO2−-N L−1, ideally for the downstream anammox in case of residual ammonium levels of around 5 mg N L−1. Since Thiobacillus members play a key role in managing nitrite accumulation, their abundance should be guaranteed in the practical application.

Elemental sulfur autotrophic partial denitrification (S0-PDN) with high pH and free ammonia control strategy for low-carbon wastewater: From performance to microbial mechanism

Elemental sulfur autotrophic partial denitrification (S0-PDN) with high pH and free ammonia control strategy for low-carbon wastewater: From performance to microbial mechanism

Influence of pH and dissolved oxygen control strategies on the performance of pilot-scale microalgae raceways using fertilizer or wastewater as the nutrient source

Dissolved oxygen concentration and pH are controllable and cost-effective variables that determine the success of microalgae-related processes. The present study compares different control strategies for pH and dissolved oxygen in pilot-scale microalgae production systems. Two 80 m2 raceway reactors were used, one operated with freshwater plus fertilizer and the other with wastewater as the nutrient source. Both were in semi-continuous mode at a fixed dilution rate of 0.2 day−1. A comparison between the classical On-Off and more advanced pH control strategies, such as PI and Event-based control, was performed, focusing on biomass productivity and the influence of all the process parameters on microalgae growth; "No control" of pH was also assayed. The results show that Event-based control was the best algorithm when using freshwater plus fertilizer. In contrast, no significant differences were observed using the different control strategies when wastewater was the nutrient source. These experiments were performed through selective control strategy, prioritizing pH over dissolved oxygen; however, it was demonstrated that they did not allow to achieve satisfactory dissolved oxygen removal results, especially for the fertilizer system. After modifying the gas diffuser configuration and improving the mass transfer, independent on-off strategies have been developed, permitting effective control of both variables and increasing productivity by up to 20% in both systems. Concluding, a detailed analysis of the energy demand for each strategy implemented in terms of gas consumption and gas flow to biomass ratio is provided.

A learning-based model predictive strategy for pH control in raceway photobioreactors with freshwater and wastewater cultivation media

This article presents a study on the pH control of raceway photobioreactors (PBRs) using a learning-based model predictive control (LBMPC) approach. The primary objective is to enhance the control performance and improve microalgae biomass production. The research builds upon existing control solutions for PBRs found in the literature by integrating the robust and adaptive capabilities of the LBMPC. Specifically, the control algorithm is modified to include a pre-established Linear Quadratic Tracking with a Feedforward controller, facilitating the formulation of the conventional LBMPC. To validate the proposed controller, implementation is performed in an existing facility at the IFAPA center, located near the University of Almería in Southern Spain. The experiment involves two parallel PBRs operating under the same meteorological conditions with different microalgae strains and media (freshwater and wastewater). The LBMPC demonstrates satisfactory results and is compared with the conventional nominal MPC strategy through an analogous experiment. The LBMPC outperforms the conventional approach, achieving up to four times superior performance in terms of the average error index. The results highlight the importance of employing robust adaptive control strategies for highly nonlinear and multi-disturbed systems like the variant biological-chemical microalgae process in PBRs since the complexity associated with adapting the system’s model can negatively impact the performance of conventional model-based controllers.

Establishment of picodroplet-based co-culture system to improve erythritol production in Yarrowia lipolytica

Fluorescence-activated droplet sorting (FADS) combined with transcription factor (TF)-based biosensors allows for the high-throughput isolation of clones that overproduce extracellular metabolites. Here, we developed a FADS-TF coculture pipeline for improving erythritol production in Yarrowia lipolytica. The pipeline comprises three droplet operation steps: the generation and incubation of droplets that encapsulate yeast mutants with single-cell resolution, the pico-injection of fluorescence-based erythritol-biosensing Escherichia coli, and the sorting of the highest fluorescence droplets with an approximately 1 ‰ survival ratio. With the picodroplet-based workflow, we initially carried out a two-stage temperature and pH control strategy on the droplets to separate the erythritol production and detection processes. We then eliminated the background expression of biosensors by utilizing pH-sensitive erythromycin, finally establishing a complete version of the coculture screening system. The optimal strain, Yarrowia lipolytica S4-9, contributed 231.2 g/L erythritol after a 114-h fermentation and was successfully screened from the fourth round of the mutant library. Compared to the parent, the erythritol yield and production rate of Yarrowia lipolytica S4-9 increased by 16.97 % and 26.09 %, respectively, in a 5-L bioreactor. Our results indicated that FADS-TFs have great potential for the high-throughput screening of extracellular products as additional biosensors become available.

Integrated biohydrogen production and dairy manure wastewater treatment via a microalgae platform

The present study aimed to investigate the feasibility of sustainable biohydrogen production from dairy manure wastewater using a microalgae platform to achieve simultaneous wastewater treatment and clean energy production. Compared to single-stage microalgae cultivation, two-stage cultivation of indigenous wastewater bacteria and carbohydrate-rich microalgae Chlorella vulgaris ESP-6 achieved satisfactory removal of nutrients and chemical oxygen demand. The resulting biomass, with high carbohydrate content (48.8 ± 2.3%), was used as alternative feedstock for biohydrogen production by Clostridium butyricum CGS5 via dark fermentation after thermal acid pretreatment. The implementation of an automatic pH control strategy significantly promoted H2 generation, resulting in a hydrogen yield of 211 mL/g VS and a maximum productivity of 391.1 mL/L/h. To the best of our knowledge, this is the first integrated study of dairy manure wastewater treatment and biohydrogen production with high H2 yield and productivity comparable to those reported in the literature.

Biosynthesis of Gamma-Aminobutyric Acid by Engineered Clostridium tyrobutyricum Co-Overexpressing Glutamate Decarboxylase and Class I Heat Shock Protein

Gamma-aminobutyric acid (GABA) is a major inhibitory neurotransmitter in the mammalian central nervous system that has a significant beneficial effect on human health. Traditional microbial GABA synthesis requires continuous oxygen supplementation. Here, a new anaerobic platform for GABA production was established with engineered C. tyrobutyricum ATCC 25755, which is considered an ideal anaerobic microbial-cell factory for bioproduction. Glutamate decarboxylase (GAD) and Class I heat-shock proteins were screened and overexpressed, generating an excellent Ct-pMAG strain for monosodium-glutamate (MSG) tolerance and GABA production, with a GABA titer of 14.26 g/L in serum bottles with the mixed substrate of glucose and MSG. Fed-batch fermentation was carried out in a 5 L bioreactor, achieving 35.57 g/L and 122.34 g/L final titers of GABA by applying the pH-free strategy and the pH-control strategy, respectively using MSG. Finally, a two-stage strategy (growth stage and bioconversion stage) was applied using glutamate acid (L-Glu) and glucose as the substrate, obtaining a 400.32 g/L final titer of GABA with a productivity of 36.39 g/L/h. Overall, this study provides an anaerobic-fermentation platform for high-level bio-GABA production.

A Light Scattering Investigation of Enzymatic Gelation in Self-Assembling Peptides.

Self-assembling peptides (SAPs) have been increasingly studied as hydrogel-former gelators because they can create biocompatible environments. A common strategy to trigger gelation, is to use a pH variation, but most methods result in a change in pH that is too rapid, leading to gels with hardly reproducible properties. Here, we use the urea-urease reaction to tune gel properties, by a slow and uniform pH increase. We were able to produce very homogeneous and transparent gels at several SAP concentrations, ranging from c=1g/L to c=10g/L. In addition, by exploiting such a pH control strategy, and combining photon correlation imaging with dynamic light scattering measurements, we managed to unravel the mechanism by which gelation occurs in solutions of (LDLK)3-based SAPs. We found that, in diluted and concentrated solutions, gelation follows different pathways. This leads to gels with different microscopic dynamics and capability of trapping nanoparticles. At high concentrations, a strong gel is formed, made of relatively thick and rigid branches that firmly entrap nanoparticles. By contrast, the gel formed in dilute conditions is weaker, characterized by entanglements and crosslinks of very thin and flexible filaments. The gel is still able to entrap nanoparticles, but their motion is not completely arrested. These different gel morphologies can potentially be exploited for controlled multiple drug release.

Improving the Electron Transport Performance of TiO2 Film by Regulating TiCl4 Post-Treatment for High-Efficiency Carbon-Based Perovskite Solar Cells.

Titanium oxide (TiO2 ) has been widely used as an electron transport layer (ETL) in perovskite solar cells (PSCs). Typically, TiCl4 post-treatment is indispensable for modifying the surfaces of TiO2 ETL to improve the electron transport performance. However, it is challenging to produce the preferred anatase phase-dominated TiO2 by the TiCl4 post-treatment due to the higher thermodynamic stability of the rutile phase. In this work, a mild continuous pH control strategy for effectively regulating the hydrolysis process of TiCl4 post-treatment is proposed. As the weak organic base, urea has been demonstrated can maintain a moderate pH decrease during the hydrolysis process of TiCl4 while keeping the hydrolysis process relatively mild due to the ultra-weak alkalinity. The improved pH environment is beneficial for the formation of anatase TiO2 . Consequently, a uniform anatase-dominated TiO2 surface layer is formed on the mesoporous TiO2 , resulting in reduced defect density and superior band energy level. The interfacial charge recombination is effectively suppressed, and the charge extraction efficiency is improved simultaneously in the fabricated solar cells. The efficiency of the fabricated carbon electrode-based PSCs (C-PSCs) is improved from 16.63% to 18.08%, which is the highest for C-PSCs based on wide-bandgap perovskites.

Biogas upgrading in a pilot-scale trickle bed reactor – Long-term biological methanation under real application conditions

The biological methanation of H2 and CO2 in trickle bed reactors is one promising energy conversion technology for energy storage, but experiences at pilot-scale under real application conditions are still rare. Therefore, a trickle bed reactor with a reaction volume of 0.8 m3 was constructed and installed in a wastewater treatment plant to upgrade raw biogas from the local digester. The biogas H2S concentration of about200 ppm was reduced by half, but an artificial sulfur source was required to completely satisfy the sulfur demand of the methanogens. Increasing the ammonium concentration to > 400 mg/L was the most successful pH control strategy, enabling stable long-term biogas upgrading at a CH4 production of 6.1 m3/(m3RV·d) with synthetic natural gas quality (CH4 > 98%). The results of this study with a reactor operation period of nearly 450 days, including two shutdowns, represents an important step towards the necessary full-scale integration.

Adaptive evolutionary strategy coupled with an optimized biosynthesis process for the efficient production of pyrroloquinoline quinone from methanol

BackgroundPyrroloquinoline quinone (PQQ), a cofactor for bacterial dehydrogenases, is associated with biological processes such as mitochondriogenesis, reproduction, growth, and aging. Due to the extremely high cost of chemical synthesis and low yield of microbial synthesis, the election of effective strains and the development of dynamic fermentation strategies for enhancing PQQ production are meaningful movements to meet the large-scale industrial requirements.ResultsA high-titer PQQ-producing mutant strain, Hyphomicrobium denitrificans FJNU-A26, was obtained by integrating ARTP (atmospheric and room‑temperature plasma) mutagenesis, adaptive laboratory evolution and high-throughput screening strategies. Afterward, the systematic optimization of the fermentation medium was conducted using a one-factor-at-a-time strategy and response surface methodology to increase the PQQ concentration from 1.02 to 1.37 g/L. The transcriptional analysis using qRT-PCR revealed that the expression of genes involved in PQQ biosynthesis were significantly upregulated when the ARTP-ALE-derived mutant was applied. Furthermore, a novel two-stage pH control strategy was introduced to address the inconsistent effects of the pH value on cell growth and PQQ production. These combined strategies led to a 148% increase in the PQQ concentration compared with that of the initial strain FJNU-6, reaching 1.52 g/L with a yield of 40.3 mg/g DCW after 144 h of fed-batch fermentation in a 5-L fermenter.ConclusionThe characteristics above suggest that FJNU-A26 represents an effective candidate as an industrial PQQ producer, and the integrated strategies can be readily extended to other microorganisms for the large-scale production of PQQ.

Machine learning and metabolic modelling assisted implementation of a novel process analytical technology in cell and gene therapy manufacturing

Process analytical technology (PAT) has demonstrated huge potential to enable the development of improved biopharmaceutical manufacturing processes by ensuring the reliable provision of quality products. However, the complexities associated with the manufacture of advanced therapy medicinal products have resulted in a slow adoption of PAT tools into industrial bioprocessing operations, particularly in the manufacture of cell and gene therapy products. Here we describe the applicability of a novel refractometry-based PAT system (Ranger system), which was used to monitor the metabolic activity of HEK293T cell cultures during lentiviral vector (LVV) production processes in real time. The PAT system was able to rapidly identify a relationship between bioreactor pH and culture metabolic activity and this was used to devise a pH operating strategy that resulted in a 1.8-fold increase in metabolic activity compared to an unoptimised bioprocess in a minimal number of bioreactor experiments; this was achieved using both pre-programmed and autonomous pH control strategies. The increased metabolic activity of the cultures, achieved via the implementation of the PAT technology, was not associated with increased LVV production. We employed a metabolic modelling strategy to elucidate the relationship between these bioprocess level events and HEK293T cell metabolism. The modelling showed that culturing of HEK293T cells in a low pH (pH 6.40) environment directly impacted the intracellular maintenance of pH and the intracellular availability of oxygen. We provide evidence that the elevated metabolic activity was a response to cope with the stress associated with low pH to maintain the favourable intracellular conditions, rather than being indicative of a superior active state of the HEK293T cell culture resulting in enhanced LVV production. Forecasting strategies were used to construct data models which identified that the novel PAT system not only had a direct relationship with process pH but also with oxygen availability; the interaction and interdependencies between these two parameters had a direct effect on the responses observed at the bioprocess level. We present data which indicate that process control and intervention using this novel refractometry-based PAT system has the potential to facilitate the fine tuning and rapid optimisation of the production environment and enable adaptive process control for enhanced process performance and robustness.

Effluent pH correlates with electrochemical nitrogen recovery efficiency at pilot scale operation

A bipolar electrodialysis (BP-ED) pilot plant including 3.15 m2 of cation exchange membrane and bipolar membrane each was operated for ammonia recovery. The pilot treated source-separated diluted urine (1 gNH4+/L). Previously found set operation parameters for lab-scale such as current density and nitrogen load did not directly influence the stack performance. However, the effluent pH was directly related to the removal efficiency of the system. 80% nitrogen removal was achieved at a set effluent pH of 4. Operating under an effluent pH control strategy was more effective to control NH4+ removal than controlling current density or nitrogen loading, as it accounts for fluctuation in wastewater availability and composition. The pilot plant removed up to 88% of the NH4+ from urine and recovered around 700 g/day (from 1 m3 of urine). This was a significant improvement compared to the pilot plant previous performance on digestate. The energy consumption was around 13 Wh/gN. The overall current efficiency was ∼40% with most losses caused by parasitic ionic shortcut currents occurring at the hydraulic manifolds of the BP-ED stack. Therefore, the energy demand can be further decreased by preventing these ionic shortcuts in the new cell designs.

Synergy of selective buffering, intermittent pH control and bioreactor configuration on acidogenic volatile fatty acid production from food waste.

The production of volatile fatty acids (VFAs) and biohydrogen (bio-H2) from food waste (FW) by acidogenic process is one of the promising strategies. The present study was performed to evaluate the role of initial (phase I) and intermittent pH (phase II) control strategies utilising combination of sodium hydroxide (NaOH) and sodium carbonate (Na2CO3) as buffering/neutralizing agents on VFAs and bio-H2 production from FW. The study was carried out in two bioreactor configurations (biofilm (UAFBB) and a suspended mode bioreactor (UASB)). Intermittent pH adjustment (phase II) increased hydrolysis and FW acidification compared to the initially adjusted pH (phase I), but had a detrimental influence on bio-H2 generation in both the studied bioreactor configurations. Combining NaOH and Na2CO3 resulted in higher buffering capacity and VFA production. The studied parameters in UAFBB aided in higher VFA (14.05g/L; 48h of cycle operation) and bio-H2 (56%; 12h of cycle operation) production during phase II and phase I operation, respectively. Overall, the results showed a synergy between the examined parameters, resulting in increased VFA production from FW.

Production of zinc protoporphyrin IX by metabolically engineered Escherichia coli.

Zinc protoporphyrin IX (ZnPPIX) has been considered as a promising red colorant for food industries as well as an anticancer drug. However, bio-based production of ZnPPIX from a renewable carbon source has not been reported yet. In this study, a fermentation process of the metabolically engineered Escherichia coli HAEM7 strain was optimized for the high-level production of ZnPPIX. To repurpose the HAEM7 strain that was originally developed for the production of heme into a producer of ZnPPIX, the concentrations of iron and zinc in the culture medium were rebalanced. Next, the concentration of zinc in the feeding solution was optimized to improve ZnPPIX production. Moreover, the pH control strategy, induction point, and the strategy of increasing the cell density, which were optimized in the accompanying paperfor the high-level production of heme, were applied together. In the optimized fermentation process, the HAEM7 strain produced 2.2 g/L ZnPPIX with a productivity of 39.9 mg/L/h. The fermentation process and strategies reported here will expedite establishing industry-level production of ZnPPIX.