Biochemical Engineering Journal最新文献

Despite extensive efforts in biotherapeutics production, there is a pressing need to enhance the biomanufacturing process to meet the growing demand for recombinant products. Ubiquitous Chromatin Opening Element (UCOE), chromatin-modifying elements derived from CpG islands, are crucial in regulating gene expression. Therefore, to explore the UCOE effect on gene expression, we cloned codon-optimized Darbepoetin alfa (DPO) sequence into the pOptiVEC then the Darbepoetin alfa-IRES-DHFR fragment was digested, and cloned into the UCOE vector. Both pOptiVEC-DPO and UCOE-DPO-IRES-DHFR cassettes were linearized and transfected into the two cell pools of CHO DG44 cells. The transfected cells were adapted with CD OptiCHO™ medium after 20 days. Recombinant DPO expression was evaluated by qRT-PCR technique. Qualitative and quantitative protein expression analysis was also carried out by western blotting and ELISA assays. Our findings showed a boosted DPO expression rate at both mRNA and protein levels in the UCOE-DPO-IRES-DHFR pool compared to pOptiVEC-DPO. These data suggest that UCOE and codon optimization result in high and stable gene expression in the CHO DG44 cell line.

The lack of denitrifying bacteria and organic carbon sources, and the inhibition of dissolved oxygen (DO) result in nitrate accumulation in aquaculture wastewater. In order to solve this problem, an encapsulation method was introduced to prepare a novel bioactive capsule, which can provide organic carbon source, denitrifying bacteria, and anoxic microenvironment for aquaculture wastewater denitrification. And can reduce the recovery time of the enclosed denitrifying bacteria. The morphology of the capsule, its nitrate removal rate, and nitrogen conversion pathway in synthetic aquaculture wastewater were investigated. The capsule had a porous surface and the pore diameter ranged from 150.0 nm to 300.0 nm. The enclosed denitrifying bacteria had a reduced recovery time and excellent denitrification performance. The nitrate removal rate reached 86.2 % on the first day and was maintained at 99.7 %. Nitrogen conversion pathways in the capsule include denitrification, assimilatory/dissimilatory nitrate reduction, and nitrogen fixation. The denitrifying capsule has short recovery time and good denitrification performance, which would help to achieve denitrification in aquaculture wastewater or other low C/N wastewater.

This study examined the impact of different biochar (BC) as an anaerobic digestion (AD) additive on antibiotic-resistant bacteria (ARB) survival and AD performance using dairy cow manure. Bamboo BC and Olive BC with different particle sizes were added into the mesophilic AD at 15 g/L and 30 g/L dosages (Bamboo-15, Bamboo-30, Olive-15, and Olive-30). The study provides a detailed analysis of biogas production, organic metabolism, and ARB and microbial dynamics, elucidating the mechanisms by which BC influences AD. Findings reveal significant reductions in CEZ-resistant bacteria (CEZ-r) across all reactors, ranging from 12.88 % to 76.47 %. Both Bamboo and Olive BC increased CEZ-r removal by 3.08–5.94 times compared to the control. Additionally, BC supplementation prevented the rise in CEZ-r percentage within the total bacteria count observed in the control reactor. Bamboo BC outperformed Olive BC in enhancing biogas yield, with Bamboo-15 and Bamboo-30 showing significant increases of 43.2 % and 48.0 %, respectively, compared to the control. Adding BC in AD regulates ARB by decreasing potential ARG hosts and impeding the transmission of resistance. It also enhances biogas production by improving the efficiency of methanogenic bacteria and optimizing the methanogenic pathway. This research provides insights into how BC can be used to enhance AD performance and mitigate ARB proliferation, offering a sustainable approach to waste management and energy production.

Longan (Dimocarpus longan Lour.) is a nutrient-rich fruit, widely cultivated in China. However, its seeds and pericarp, often discarded as waste, are rich in bioactive components, particularly polyphenolics. In this research, we develop microbiota-assisted extraction technique, which is efficient and environmentally friendly for extracting these valuable components. These results showcased superior efficiency in the diversity and richness of extracted bioactive components, although yielding slightly lower TP content than ethanol extraction. The active components extracted by microbiota-assisted extraction technique increased to 2.45 mg/g, which was higher 2.13 times and 3.10 times compared to optimized ethanol and water extraction, respectively. The numbers of active components extracted by these three methods were 39, 24 and 30, separately, and Quercetin 3-D-xyloside and Kaempferol 3-O-alpha-L-rhamnopyranosyl were identified in longan pericarp extracts by microbiota-assisted extraction. Metagenomics analysis revealed stable and diverse microbiota with functional capabilities for enhancing extraction efficiency. Functional annotations indicated significant roles in biosynthesis, carbohydrate degradation, and fatty acid metabolism. Microbiota-assisted extracts exhibited a broad spectrum of bioactive compounds with potential health benefits, including antioxidant and anti-inflammatory properties. These results provide a theoretical foundation for developing sustainable extraction technologies.

Significant progress has been achieved in large-scale mammalian cell culture technology for biotherapeutics manufacturing over the past decades, necessitating the Process Analytical Technology (PAT) for the real-time measurement of critical quality attributes and the guidance for precise process control to ensure productivity, quality, and consistency. The Oxygen Uptake Rate ($\mathrm{OUR}$) serves as a crucial indicator for characterizing the energy metabolism of mammalian cells, offering insights into cellular state and metabolism dynamics. However, current cellular $\mathrm{OUR}$ monitoring in antibody production depends mainly on costly gas analyzers or periodic manual sampling. Here, we introduce a novel method for in-line monitoring of cellular $\mathrm{OUR}$ in bioreactors based on the stationary liquid phase balance (SLPB) theory, which extends its applicability to diverse aeration and foam conditions without additional equipment or labor expenditures. We modeled the $k_{L}a$ of the aerated stirred bioreactor, assessed the influence of foam on liquid surfaces induced by gas sparging on oxygen transfer, and processed raw $\mathrm{OUR}$ data using a sliding filter. The established method was applied to monitoring the real-time $\mathrm{OUR}$ of Chinese Hamster Ovary (CHO) cell cultures for antibody production, demonstrating its excellent accuracy, sensitivity and readability. Aligned with the Quality by Design (QbD) concept, this real-time $\mathrm{OUR}$ estimation enables rapid detection of metabolic changes, revealing cellular physiology and facilitating precise feedback control in biotherapeutics manufacturing.

Complex bispecific antibody formats tend to form more product-related impurities than monoclonal antibodies. The primary constraints are yield and purity in the polishing stage. The purpose of this study was to enhance the understanding of the optimal working window for four mixed-mode resins and to reduce the burden of resin screening and parameter optimization during process development. This study optimized the loading and elution conditions of four different mixed-mode cationic resins to enhance the yield and purity by integrating Design of Experiments with High-Throughput Screening. It was observed that despite being weakly acidic mixed-mode cationic resins, these four resins exhibited significant differences in their adsorption and elution performances and varied tolerances to salt concentrations. Capto MMC demonstrated strong hydrophobicity, while the performance profiles of MX-Trp-650 M and Nuvia cPrime were similar, with Nuvia cPrime showing superior purification effects. Eshmuno CMX, by extending its side chain ligand, achieved higher binding efficiency and capacity. Through the optimization of salt concentrations or the application of dual-gradient elution strategies, the target protein with high yield (77 %) and purity over 99 % was successfully obtained. This research not only provides in-depth insights into the application of mixed-mode chromatography in the biopharmaceutical field but also offers practical optimization strategies for the industrial-scale purification of bispecific antibodies.

Bacillus velezensis P#02 simultaneously produced surfactin and poly–γ–glutamic acid (γ–PGA). Among the different culture media studied, the one containing corn steep liquor (100 mL/L), glucose (10 g/L), and glutamic acid (10 g/L) as sole ingredients (CSL–G–Glut(10)) offered the best results regarding biosurfactant and biopolymer production. Although biosurfactant production occurred both under shaking and static conditions, significant biopolymer production occurred only in static cultures. Using the culture medium CSL–G–Glut(10), 910 ± 20 mg surfactin/L and 9.8 ± 0.2 g γ–PGA/L were produced. Surfactin was synthetized as a mixture of five different homologues (fatty acid chains ranging between C₁₂ and C₁₆), being the most abundant C₁₄– and C₁₅–surfactin. Surfactin reduced the surface tension up to 29 mN/m, with a critical micelle concentration of 52 mg/L, and exhibited a significant emulsifying activity. B. velezensis P#02 γ–PGA, which molecular weight was around 229 kDa, displayed a non–Newtonian shear–thinning profile, achieving apparent viscosity values around 3800 mPa s in aqueous solution, with a predominant viscous behavior. Accordingly, B. velezensis P#02 is a promising strain for the simultaneous production of γ–PGA and surfactin using the waste stream corn steep liquor.

Aromatic amines, the common organic metabolites of chemical raw materials and herbicides, has attracted wide attention due to its difficult degradation and carcinogenic risk. This study aims to use microbial co-metabolism technology to efficiently degrade p-chloroaniline (PCA), which is a highly toxic aromatic amine. From the perspective of enzyme substrate specificity, a system for efficient degradation of PCA using aniline as a co-substrate was constructed. The degradation conditions were optimized by response surface methodology, and the degradation efficiency of PCA was 81.12 % (50 mg/L). Further, the co-metabolism mechanism was clarified by multiple methods. Enzyme activity assay preliminarily showed that aniline induced catechol 2,3-dioxygenase activity. Then the intermediates of PCA and aniline degradation was identified and two possible PCA degradation pathways were proposed. Transcriptomic analyzed the molecular mechanism of aniline-enhanced PCA degradation: Nitrogen utilization efficiency was accelerated by up-regulation of nitrogen metabolism-related genes. Several oxidoreductases including catechol 2,3-dioxygenase were significantly up-regulated. TCA cycle and ATP synthesis were accelerated, facilitating cell metabolism and energy supply. The work contributes a worthy theory for the remediation of PCA-aniline co-contaminated sites.

Pyruvate, a pivotal metabolite in the glycolytic pathway, typically confronts substantial barriers to its natural accumulation within microbial cells. This study successfully facilitated the natural accumulation of pyruvate in Actinobacillus succinogenes 130Z by fine-tuning the oxidation-reduction potential (ORP) in the fermentation milieu. A mechanistic exploration revealed that the accumulation of pyruvate was optimized when ORP conditions favorably modulated pyruvate kinase activity and concurrently suppressed succinate dehydrogenase activity. By integrating the influence of metal ions on enzymatic functions with an innovative aluminum ion-mediated ORP control strategy, we achieved a pyruvate yield of 27.54 g/L over 20 hours, which constitutes an 89.54 % increase compared to the baseline. Additionally, the production rate of pyruvate reached 1.38 g/L·h. This investigation not only elucidates the metabolic underpinnings that facilitate the natural enrichment of glycolytic intermediates in Actinobacillus succinogenes 130Z but also lays a robust theoretical foundation for the industrial-scale fermentation of pyruvate. Moreover, the capability to efficiently and rapidly concentrate essential platform metabolites within the glycolytic pathway is of paramount significance, potentially propelling forward the research and synthesis of various downstream metabolic products.

To improve heat tolerance and biomass yield of microalgae cells cultivated with flue gas in power plants in South China in summer, Scenedesmus quadricauda cells were cultivated at various temperatures to regulate functional metabolic pathways. The microalgae biomass production was 26 % higher at 35°C than at 25°C. The expression of photosynthesis-related proteins was up-regulated by 14.3 %, enhancing electron transfer efficiency and oxygen release rate at photosynthetic carbon fixation. Furthermore, microalgal cells absorbed more sulfur to enhance sulfur metabolism. The extracellular polymeric substances (EPS) content increased by 2.71-fold, improving the survival activity under high-temperature stress. The up-regulation of lysosomes and hydrogenases promoted the cellular removal of metabolic wastes and damaged organelles and improved the antioxidant defense capacity. Moreover, the microalgal cells maintained normal growth at 40°C through a self-regulatory mechanism. In contrast, the photosynthetic carbon fixation of microalgae cells was strongly inhibited at 42°C. This study revealed the adaptive mechanism of cellular carbon fixation in microalgae at high temperatures, which improved the high-temperature tolerance and biomass production of microalgae.