Bioprocess and Biosystems Engineering最新文献

Electrospun nanofibers have attracted significant interest due to their high surface area-to-volume ratio, porosity, interconnected voids, and advantageous mechanical, chemical, and physical properties. Enzymes, known for its exceptional catalytic properties, are promising candidates for various industrial applications. However, the use of free enzymes is limited by challenges such as poor recyclability and susceptibility to environmental factors. Immobilization techniques offer a viable solution by enhancing the stability and activity of enzymes. This review compares four enzyme immobilization methods to identify the most effective strategy and focuses on the various approaches to optimize electrospinning methods, as well as parameters to maximize enzyme loading, activity retention, and stability. Among the various immobilization methods, entrapment and encapsulation of enzymes within electrospun nanofibers have garnered significant attention in recent years. The review discusses the applications and challenges associated with enzyme entrapment and encapsulation using electrospinning. Overall, advancements in electrospun nanofibers with encapsulated or entrapped enzymes highlight their potential as robust, efficient, and sustainable platforms for biosensors, therapeutics, antimicrobial applications, smart textiles, as well as food and wastewater treatment processes. Subsequently, future research should focus on scalable electrospinning processes, the development of eco-friendly materials, long-term enzyme stability, multi-enzyme systems, and a deeper mechanistic understanding to further enhance performance and safety.

Production of Fab (fragment antigen-binding) molecules using Escherichia coli as a host presents a significant challenge due to low protein expression and the resulting poor yields. In this study, recombinant Ranibizumab was expressed in E. coli as inclusion bodies (IB) and optimization of lysis parameters, IB recovery, and IB washing conditions was performed to achieve optimal product yield and purity. Design of experiments (DOE) was employed to explore the interaction between variables and to facilitate optimization of buffer composition. Optimization of lysis buffer resulted in a yield of 0.069 g protein/g IB, 61% IB purity, and 87% lysis efficiency. Optimization of homogenization conditions, using two passes at 1000 bar, resulted in a 93.5% lysis efficiency with 60% IB purity. Additionally, optimizing the IB washing steps with 1% Triton X-100 and 2 M urea for 30 min at room temperature offered 84.53% IB recovery and 75% IB purity. Further, the impact of IB quality on refolding yield has been examined. Overall, the process optimization translated into a significant improvement in refolding yield, which increased from 18% under unoptimized conditions to 29% post-optimization and it has been demonstrated that optimization of lysis and washing steps can significantly enhance refolding yield, a key hurdle when expressing Fabs in E. coli.

Despite many reports focusing on the engineering aspects of biodesulfurization, there is a lack of comprehensive analysis on metabolic pathways and integration of engineering and metabolism. In this study, a genome-scale metabolic model was reconstructed for Thioalkalivibrio versutus D301, a potent strain in biodesulfurization. The model, named TVD301, was refined using extracted RNA sequencing data, and flux balance analysis demonstrated its accuracy in predicting growth and sulfur species rates. Importantly, experimental validation in a regulated medium confirmed a 60% decrease in sulfate production compared to control cultures, showing the strong practical relevance of the model. The TVD301 model also revealed that T. versutus lacks the enzymes needed to convert sulfide to sulfate, making it a strong strain in biodesulfurization. To optimize sulfur recovery and reduce sulfate production in industrial processes using microbial consortia, the TVD301 model was adapted to a consortium model. Sensitivity analysis highlighted the importance of DsrAB and Cys enzymes in preventing undesired sulfate production. By inhibiting these enzymes via inhibitors extracted from Brenda database, elemental sulfur production increased significantly. These findings suggest promising strategies for enhancing biodesulfurization processes in industrial settings.

S-adenosylmethionine (SAM) is a high-value metabolite with widespread applications in medicine and nutrition, yet its microbial production remains constrained by high energy demands and inefficient precursor utilization. In this study, we investigated sodium citrate supplementation as a strategy to enhance SAM biosynthesis in Pichia pastoris under methanol induction. Integrating transcriptomics with a newly reconstructed genome-scale metabolic model (iLD1283), we systematically elucidated the molecular and metabolic mechanisms underlying citrate-mediated improvements. Physiological analysis revealed that sodium citrate supplementation significantly increased biomass accumulation, methanol and L-methionine assimilation, and intracellular ATP levels, resulting in a 70% enhancement in SAM titer. Transcriptomic profiling demonstrated global metabolic reprogramming, including the upregulation of glycolysis, the tricarboxylic acid cycle, oxidative phosphorylation, and amino acid biosynthesis, collectively supporting improved energy supply and precursor availability. Constraint-based simulations using iLD1283 identified an optimal citrate feeding rate that balanced energy generation and SAM production, which was validated in 5-L fed-batch fermentation, achieving a peak SAM concentration of 10.87 g/L. Metabolic flux analysis further confirmed increased flux through central carbon pathways and elevated cofactor regeneration. Together, these findings provide mechanistic insight into sodium citrate-induced metabolic rewiring and establish a model-guided framework for rational optimization of energy-intensive microbial processes. This work highlights the potential of combining omics data and metabolic modeling to guide precision feeding strategies for enhanced bioproduction.

The rising demand for spirulina (Limnospira spp.) highlights the need for affordable cultivation methods and practical biomass monitoring solutions. This study introduces a novel, low-cost, Raspberry Pi-based system for real-time monitoring and automated biomass recovery in microalgal cultivation. The system integrates turbidity, light, pH, and temperature sensors with an automated module for harvesting and medium replenishment. Cultures of the filamentous, spiral-shaped microalga Limnospira fusiformis were used to evaluate system performance. The turbidity sensor showed strong correlation with optical density (R² = 0.943-0.986, p < 0.05) and dry weight (R² = 0.954-0.975, p < 0.05). Light, pH, and temperature sensors demonstrated average percentage errors of 0.50%, 0.58%, and 2.52%, respectively (p < 0.05). The auto-recovery system successfully maintained biomass concentration within a narrow range (OD₇₅₀ = 0.67-0.74) using adjustable set points tailored to cultivation needs. Real-time data were auto-logged to Google spreadsheets for remote access. With an estimated cost of $340, the system offers a practical, time-saving, and cost-effective solution for real-time biomass monitoring and control in microalgae cultivation.

Polyhydroxyalkanoate (PHA) is a bioplastic attracting interest as an alternative to petroleum-based plastics. Particularly, poly (3-hydroxybutyrate-co-3-hydroxyhexanoate) (P(3HB-co-3HHx)), which shows notable polymeric properties, is usually produced using the engineered Cupriavidus necator. Currently, production of P(3HB-co-3HHx) is primarily possible by engineering phaC, however, relatively rare study of controlling the expression of enoyl-CoA hydratase (phaJ_Pa), which is directly involved in 3-hydroxyhexanoate (3HHx) monomers synthesis, was shown to control 3HHx mole fraction. As a result, we aimed to verify this by constructing vectors housing phaC_BP-M-CPF4 and phaJ_Pa with different ribosome-binding site (RBS) to control PhaJ translation. When different constructions were applied, the fluctuation in the 3HHx molar fraction was directly related to the phaJ_Pa RBS sequence and it was shown that varying the RBS sequence to AAAGGAGATATAG produces increased 3HHx mole fraction (3.6-6.2%). When fermentation was performed for 168 h to verify the capacity of the engineered strain (H16/pSJ-3) for mass production, it produced 194.9 g/L dry cell weight and 155.4 g/L of P(3HB-co-9.5 mol% 3HHx). Overall, this study presents a different approach of altering polymer properties for manipulating the 3HHx mole fraction of P(3HB-co-3HHx) by controlling PhaJ translation.

C-Phycocyanin (C-PC), a fluorescent photosynthetic protein derived from cyanobacteria, is used in the food, cosmetic, pharmaceutical, and biotechnology industries. Various cyanobacterial sources of C-PC have been studied to harness its biological functions such as antimicrobial, antioxidant, anticancer, and anti-inflammatory properties. Phormidium sp. A02 isolate from the Indian coast was cultured in a mixotrophic static environment to determine the effect of various bioprocess parameters like culture medium and light (photoperiod, light intensity, and light color) on biomass productivity and C-PC content. The C-PC from Phormidium sp. A02 can be used in the food and cosmetic industry as an alternative to synthetic chemical colorants. Carbon-mediated metabolic engineering of C-PC in Phormidium sp. A02 using Guillard's F/2 seawater medium supplemented with carbon sources like glucose, sucrose, glucose + peptone, and sucrose + peptone was carried out to determine its growth and C-PC enhancement efficiency. Sucrose + peptone with C/N ratio 4.76 increased Phormidium sp. A02 biomass productivity (0.197 ± 0.02 g dry weight L^-1 day^-1) by twofold compared to the autotrophic control (0.105 ± 0.01 g dry weight L^-1 day^-1). An analysis of C-PC content enhancement with glycerol supplementation showed that 0.9 g of glycerol L-1 was the optimal concentration. Higher biomass productivity (0.176 ± 0.01 g L^-1 day ^-1) was observed in photoperiods of 8/16 h light/dark and higher C-PC content (69.91 ± 4.86 mg g^-1) at lower light intensity in Phormidium sp. A02 under mixotrophic conditions. A two-phase static culture strategy was developed, beginning with 5 days of initial biomass production under white light, followed by 3 days of C-PC enhancement under monochromatic light. The dry biomass production in sucrose + peptone under white, green, and red light was similar in our two-phase static culture strategy, averaging 0.27 g L^-1. In contrast, red light induction increased C-PC more than other lights and by 6.5-fold (52.30 ± 0.002 mg g^-1) over a control with white light (7.76 ± 0.58 mg g^-1). C-PC had thermal stability up to 55 °C, pH stability up to 4.00 and a purity of 0.69. Phormidium sp. A02 cultured in a closed system under bioprocess strategies such as red-light induction, glycerol supplementation, and metabolism switchover could enhance C-PC and make it a viable culture technique.

In large-scale bioprocesses, gradients in pH, dissolved oxygen level (DO), and substrate concentrations can decrease bioprocess efficiency. Scale-down bioreactors, be it single stirred-tank bioreactors with a special feeding regime, multi-compartment bioreactors, or combinations of bioreactors, offer a promising lab-scale solution for comprehending these gradients, as they allow adjustment of gradients without incurring high costs. However, critical challenges arise when transitioning from large-scale to scale-down bioreactors. Chief among these is realistically approaching the gradient conditions of large-scale bioreactors and choosing appropriate scale-down bioreactor configurations. This review paper begins by addressing the gradients encountered in large-scale bioreactors. Afterward, various types of scale-down bioreactors are characterized and compared, highlighting their advantages and disadvantages. The suitability of scale-down bioreactors is analyzed by examples of bioprocesses with different microorganisms and mammalian cells to underscore the complexities inherent in scale-down bioprocesses and emphasize the influence of cellular responses to these conditions. Finally, the potential of miniaturized and microfluidic bioreactors is briefly discussed for future application in scale-down studies.

Microbial fuel cells (MFCs) have been proven to be a green technology for solving energy crises, but their low power density limits their large-scale practical applications. In this paper, a three-dimensional porous composite hydrogel polyvinyl alcohol/polypyrrole (PVA/PPy) with good biocompatibility was prepared by temperature-field regulation via alternating cycles between low temperature (- 20 °C) and room temperature (25 °C) and used as the anode in MFC. The three-dimensional network structure of PPy nanospheres compressed by ice crystal stress exhibited excellent charge conduction capability and ion transport performance, which significantly improved the interfacial charge transfer efficiency of PVA/PPy-5 bioanode. Besides, the addition of PVA endowed the hydrogel with mechanical properties to resist the external forces. As the results, the maximum power density of PVA/PPy-5 MFC was 1521.04 mW/m², which was 1.76, 2.16 and 8.32 times higher than that of PVA/PPy-0, PPy-5 and carbon felt MFCs, respectively. Such enhancement could be attributed to the combined effects of three factors, including the FT process, biocompatibility of PVA, and bioelectrocatalytic activity of polypyrrole. The high-throughput sequencing technology revealed that the PVA/PPy-5 hydrogel anode, which facilitated the selective enrichment of electrogenic microbes, played a crucial role on the regulation of functional biofilm. This work provides a new approach for developing high-performance electrodes for MFC.

The utilization of semi-simultaneous saccharification and fermentation (SSSF) as the novel configuration has resulted in enhanced succinic acid (SA) production from lignocellulose biomass by Actinobacillus succinogenes. The effect of inoculum concentration, biomass type, substrate concentration, and fermentation configuration on SA production was examined in this study. The pre-hydrolysis process was applied to the pre-treated biomass for 6 h to facilitate the simultaneous saccharification and fermentation (SSF) process, which was then carried out for 48 h to achieve the SSSF configuration. According to the results, the production of SA from oil palm empty fruit bunch (OPEFB) through SSF and SSSF was 0.93 and 1.18 g/L and from sugarcane bagasse (SB) was 0.98 and 1.19 g/L, respectively. Results revealed, SSSF resulted in a 21-26% higher SA concentration compared to SSF. Furthermore, the concentration of the inoculum and substrate significantly affected the generation of SA from OPEFB but not for SB. According to this study, SSSF significantly enhanced SA production from lignocellulose biomass compared to SSF.