Biochemical Engineering Journal最新文献

Process intensification and media optimization, as a crucial step for improving productivity and manufacturing cost of goods (COG), set the stage for commercialization readiness and redefine the landscape for patient access. This study described a stepwise approach to explore different intensified fed-batch processes along with optimized cell culture media for the production of a Mabcalin™ bispecifics. Initially, by leveraging perfusion expansion, intensified fed-batch (IFB) with an inoculation density of 10.3 × 10⁶ cells/mL was developed to produce 6.1 g/L of products, compared to 3.9 g/L from the original traditional fed-batch (TFB). Following the IFB conversion, a high-performing production medium, MagniCHO™, was chosen to substitute the original one, which further boosted the titer to 9.1 g/L. The result underscored the significance of developing an optimized cell culture media for intensified cultivation. Furthermore, the approach of ultra-intensified intermittent-perfusion fed-batch was utilized, raising the seeding density to 73.6 × 10⁶ cells/mL. A final harvest titer of 24.5 g/L was recorded. Additionally, manufacturing COG was calculated to evaluate how process intensification could lead to improved manufacturing cost-effectiveness, with up to 71 % COG reduction attainable with the UI-IPFB process. This study demonstrated that even for difficult-to-express modalities, applying a strategic development approach including process intensification and media optimization could effectively improve manufacturing efficiency and COG competitiveness.

Rapid and accurate molecular diagnostics are crucial for disease diagnosis and precision medicine. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) proteins have emerged as highly effective tools for molecular diagnostics. Numerous nucleic acid detection instruments and biosensors utilizing the CRISPR/Cas system have been developed. The profiling activity of CRISPR/Cas effectors has facilitated the creation of instrument-free, sensitive, precise, and rapid nucleic acid diagnostics. This review summarizes recent advancements in CRISPR technology for RNA detection, focusing on the application of Cas12 and Cas13 systems in two scenarios: in combination with isothermal amplification technology and without amplification. It also explores the significant potential of CRISPR as a next-generation technology for RNA detection and anticipates future developments. The ongoing advancements in CRISPR are expected to enhance precision and convenience in RNA testing, impacting both biomedical research and public health practices.

Linalool is one of the commercially important fragrance molecule usually extracted from Lavandula angustifolia (lavender) and Ocimum basilicum (basil) plants. In the present study, efforts were made to produce this molecule in microbial system to meet demand-supply imbalance. Linalool synthase (LIS) gene from Magnolia champaca (Mc) and Coriandrum sativum (Cs) were successfully cloned and expressed in Saccharomyces cerevisiae CEN PK2–1 C. It was observed that expression of full-length LIS (fLIS) resulted in lesser linalool when compared to truncated LIS (tLIS) devoid of plastid signal for both Mc and Cs. In terms of linalool yield, MctLIS resulted in 1.27-fold higher linalool when compared to CstLIS. Later, when two more genes viz., TPI1 and ALD6 which presumably increase sterol pathway flux were overexpressed, actually resulted in lower linalool and increased acetate production. However, multicopy expression of MctLIS and tHMG1 in this strain has reversed the above phenomenon due to presumptive push-pull strategy. Finally, this engineered strain was cultivated in the 2 L bioreactor in fed-batch mode to obtain 10.85 µg/mL of linalool. Docking studies of homology model of MctLIS with geranyl pyrophosophate (GPP) revealed V387, Y361, T434, R427 and R249 as key interactions sites. The study reports the linalool production using LIS gene from Magnolia champaca for the first time and could be a potential chassis for further studies.

Kluyveromyces marxianus is a yeast capable of fermenting sugars into ethanol and growing at high temperatures (>37ºC). However, it is less tolerant to ethanol than Saccharomyces cerevisiae, which limits its application in second-generation ethanol production. Since the mechanisms of ethanol stress response are still poorly described, especially compared to S. cerevisiae, we used an integrative multi-omics approach, combining transcriptomics, co-expression networks, gene regulation, and genome-scale metabolic modelling to gain insights about these mechanisms. Through metabolic modelling, we predicted the occurrence of a respiro-fermentative metabolism and its onset as the dilution rate increased. From gene co-expression networks, we detected that the protein quality control system is a main mechanism involved in the ethanol stress response. Further, we identified key regulators in the ethanol stress response, such as HAP3, MET4, and SNF2, and assessed how disturbances in their gene expression affect cellular metabolism. We also found that amino acid metabolism, membrane lipid metabolism, and ergosterol exhibit increased metabolic flux under the explored conditions, along with usage of enzymes related to these pathways. These findings provide useful cues to develop and implement genetic and metabolic engineering strategies to enhance ethanol tolerance and point for future research in stress responses of K. marxianus.

In this study, we used microorganisms and steel slag to reduce CO₂ emissions. The main objective is to investigate the influence and mechanisms of CO₂ fixation rate based on the composition of steel slag. In the absence of microorganisms, steel slag powders with higher C₂S content exhibit higher CO₂ fixation rate. The absolute content of C₂S decreases by 2.16–5.86 % and 3.43–14.21 % at 2 h and 48 h of carbon sequestration reaction, respectively. Under the action of microorganisms, the CO₂ fixation rate of different steel slags increases by more than two-fold, with increases in amount of CO₂ fixation at 2 h and 48 h of reaction being 142–169 % and 166–191 %, respectively. Microorganisms can enhance the reaction degree of C₂S, C₃S, and C₂F phases in different steel slags. The increase in amount of CO₂ fixation is particularly significant for steel slag powders with high C₂S and C₂F content. Enzymes secreted by microorganisms in the early stage of carbon sequestration can also increase the concentration of HCO₃^- and CO₃^2- in the liquid phase, but this is influenced by the pH value and Ca²⁺ concentration of different steel slag leachates. Steel slag powders with lower leachate pH values and containing small amounts of Ca²⁺ will be more conducive to microorganisms enhancing the early-stage CO₂ fixation rate.

Cellulose acetate (CA) nanofibers have been popularly applied in various biomedical and textile products. In this work, a textile azo-dye Reactive Green 19 (RG19) was selected to be chemically coupled to the CA nanofiber membrane to form dyed CA nanofiber membrane (namely CA-RG19) and then poly(hexamethylene biguanide) (PHMB) as an antibacterial reagent was physically attached to the dyed CA nanofiber membrane, forming CA-RG19-PHMB nanofiber membrane. The nanofiber membranes were evaluated for their physical and mechanical properties, including functional group analysis, morphological characterization, and thermal stability assessment. To investigate the antibacterial properties of the nanofiber membrane, various concentrations of RG19 dye and PHMB were tested to evaluate the antibacterial efficiency (AE) against Escherichia coli of the membranes. It was found that the CA-RG19-PHMB nanofiber membrane exhibited an AE value of approximately 100 %, with the immobilization concentrations of RG19 dye and PHMB being 373.46 mg/g and 0.333 mg/g, respectively. The CA-RG19-PHMB nanofiber membrane showed 100 % antibacterial efficacy after 10 min against E. coli cells. Furthermore, the storage stability of the CA-RG19-PHMB nanofiber membrane remained at approximately 100 % of its initial antibacterial efficacy after 60 days, and it exhibited excellent antibacterial efficacy after five cycles.

Denitrification of wastewater with a low organic carbon to NO₃^--N ratio (C/N ratio) faces challenges due to slow rates and low efficiency. This study reported that carbon-based conductive carriers are able to enhance the removal of nitrogen from wastewater with low C/N ratio by coupling Fe(II)-driven autotrophic and heterotrophic bioelectrochemical denitrification. When Fe(II) was the sole electron donor, the bioreactor using conductive carrier achieved a denitrification rate constant (k_DN) of 0.016 h⁻¹, 1.7 times of that with non-conductive materials. This enhancement was due to the conductive carrier boosting direct electron transfer and supporting the growth of electroactive microorganisms. For wastewater with a low C/N ratio of 0.76, the bioreactor featuring both Fe(II) and the conductive carrier reached a k_DN of 0.095 h⁻¹, five times higher than without Fe(II). The presence of Fe(II) promoted denitrification by enhancing electron transfer and serving as a mediator. Microbial analysis showed that adding Fe(II) enriched electroactive bacteria like Comamonas and denitrifiers such as Chryseobacterium. Our findings suggest a promising strategy to enhance denitrification in wastewater treatment systems with low C/N ratios.

Mathematical models are indispensable for designing, optimizing, and controlling large-scale bioprocesses. In the present work, the production of pectinases was studied experimentally using an internal loop airlift bioreactor, pectin as a substrate, and Aspergillus flavipes FP-500 as a biocatalyst. The N-tanks-in-series (NTIS) model was implemented to predict the behavior of fungal growth, pectin and dissolved oxygen consumption, pectinases production, and the oxygen mass transfer rate from gas phase to liquid culture medium. A double Monod-Logistic kinetic model was used to describe the biomass growth rate as a function of biomass, pectin, and dissolved oxygen concentrations. In contrast, a Luedeking-Piret kinetic model was used to describe the production rate of endo and exo pectinases. A hydrodynamic model was utilized to estimate gas hold-ups, volumetric mass transfer coefficients, and air inflow velocities. Good agreement was observed between the experimental data and the theoretical results, demonstrating the predictive capacity of the NTIS model to describe pectinase production, the oxygen consumption rate, and the oxygen evolution in the gas phase. The model highlighted its robust capability to capture the critical parameters of aerobic fermentation processes. Therefore, it could be used as a tool for the scalability of the airlift bioreactors.

Cordyceps sinensis is widely known for its therapeutic properties. Enhancing the yield of exopolysaccharides (EPS), which is crucial for its medicinal efficacy, is a major challenge. In this study, we applied high initial glucose concentrations with talc particles to enhance EPS production and assessed the cell morphology, intracellular biochemical reactants, and bioactivity contribution of glycoproteins. The use of 150 g/L glucose and 10 g/L 2000 mesh talc increased the EPS yield by 1.8-fold to 4.21 g/L. The addition of talc regulated cell morphology, facilitated the entry of oxygen molecules into the cells to produce a large amount of ATP for polysaccharide synthesis, and altered the cell wall structure to facilitate the secretion of EPS. Moreover, environmental stress resulted in a notable increase in intracellular reactive oxygen species levels, which can potentially enhance cell membrane permeability and promote EPS synthesis. Furthermore, the highest protein content in crude EPS corresponded to the maximum activation of alcohol dehydrogenase (ADH) of 44.2 %, suggesting a mechanistic relationship between the proteins and polysaccharides in the glycoproteins that influence the activation of ADH. These findings elucidate the intricate interplay between fermentation conditions and EPS production and provide new avenues for optimizing the fermentation process of CS-HKI to enhance its therapeutic applications.

In this study, the effluent from recycled paper mill was treated using a combined ozone (O₃) and biological aerated filter (BAF) process. Key operational parameters such as ozone dosage, pH, hydraulic retention time (HRT), volume load and gas-to-water ratio were optimized. Under optimal conditions, with a total ozone dosage of 100 g/m, a gas-to-water ratio of 4:1, and an HRT of 3.0 hours in the BAF, the chemical oxygen demand (COD) and chroma of the treated wastewater were reduced to 44–55 mg/L and 2–4 PCU, achieving removal efficiencies of 70 % and 95 %, respectively. The discharge effluent not only satisfy the new discharge standard of China (GB3544–2008), but also can be used as recycling water. Additionally, the treatment cost of wastewater was ca. 1.3 ¥/m³ in pilot-scale test, significantly decreasing the cost. Ozone pretreatment has a significant effect on wastewater decolorization by disrupting the molecular chemical structure of pollutants, which increase the biochemical properties of biofilm and is beneficial to the sequential BAF treatment. The sludge in the O₃/BAF system exhibited increased biomass with minimal filamentous bacteria and higher dehydrogenase activity, confirming stable and robust bacterial growth. GC-MS analysis revealed substantial reduction in pollutant content and diversity post-treatment, although the recalcitrant compound (Z)-13-docosenamide remained relatively high, decreasing from 27.37 % to 21.14 %. The mechanism of the O₃/BAF process for the pollutant degradation were also proposed. This study demonstrated that a combination of ozone and fixed biofilm treatment is an efficient and cost-effective treatment, providing the theory and practical applicability for the industrial wastewater.