Bioprocess and Biosystems Engineering最新文献

Rotaviruses are the leading cause of severe diarrhea and mortality in children less than 5 years old. Vaccination is considered to be the most effective strategy to prevent rotavirus infection. Two live attenuated oral vaccines have been licensed in many countries worldwide, resulting in significant reductions in rotavirus-associated mortality and hospitalizations. However, access to these vaccines remains limited in low- and middle-income countries, partly due to their high cost of current vaccines. The spike protein VP4 of rotavirus is the main antigen responsible for inducing neutralizing antibodies. Therefore, VP4 protein could provide the antigen part of a new rotavirus subunit vaccine candidate. In this study, we developed a robust cultivation process for VP4 protein expression in Escherichia coli with yield higher than 620 mg/L and compliant with the Quality by Design approach. First, the process parameters with potential significant effect on VP4 protein yield were identify based on our experience in virus-like particle vaccine production and screened with a Fractional Factorial Design approach in 1-L parallel bioreactor system. Then, the robust setpoint and design space of the OD₆₀₀ for induction (ODI), induction temperature (ITmp), the final concentration of IPTG (Con), and speed of feed addition (SOFA) were explored based on a criteria of VP4 protein yield > 500 mg/L and probability of failure < 3%. With process parameters set at the robust setpoint, the VP4 protein yield of 685 mg/L was obtained in 1-L bioreactor. Furthermore, the VP4 protein yields with process parameters at the robust setpoint and design space vertexes were higher than 620 mg/L and within the interval of model prediction. This study may serve as a reference for development of a robust and cost-effective subunit rotavirus vaccine antigen expression process in Escherichia coli.

Low temperature can suppress biological nitrogen removal (BNR) efficiency. Although nitrogen removal (NR) characteristics of single psychrotolerant bacteria have been extensively studied, synergistic interactions between functionally distinct psychrotolerant nitrogen-removing consortia remain unexplored. In this study, a composite microbial agent, designated NDC-6, was generated by coculturing a psychrotolerant nitrifying consortium NC1 (Pseudomonas veronii HN1, P. poae HN2, and P. peli HN3) and an aerobic denitrifying consortium DC1 (Aeromonas sp. AD1, P. extremaustralis AD2, and Serratia liquefaciens AD3) at a 1:1 inoculation ratio, and its NR performance was systematically evaluated. After 3 days of incubation at 10 °C, NDC-6 achieved removal efficiencies of 89.3%, 88.1%, 85.5%, and 95.3% for NH₄⁺-N, NO₃^--N, total nitrogen (TN), and chemical oxygen demand (COD), which were significantly higher than those of individual strains or single-function consortia. Sodium succinate was identified as the optimal carbon source, which simultaneously improved biomass growth and NR efficacy of NDC-6. Optimal culture conditions determined using response surface methodology were as follows: C/N ratio, 6; temperature, 10.2 °C; pH, 7.2; and shaking speed, 156 rpm. Under these conditions, the verification experiment achieved a TN removal efficiency of 89.8%, closely approaching the theoretically predicted maximum of 90.2%. Nitrogen balance and functional gene (hao, napA, nirS, nirK, cnorB, and nosZ) expression analyses revealed that under low-temperature and aerobic conditions, NDC-6 achieved NR primarily through dual pathways of bacterial assimilation and dissimilation, converting NH₄⁺-N and NO₃^--N into intracellular nitrogen and N₂. The dissimilatory mechanism relied on synergistic metabolism and functional complementation between NC1 and DC1, mediated by their respective functional genes. This study provides mechanistic insights into the biological treatment of nitrogen-containing wastewater, particularly under low-temperature conditions, and offers a novel strategy for such treatment.

The increase of antibiotics in aquatic environments, along with the emergence of antibiotic-resistant bacteria, highlights the improvement of wastewater treatment technologies. This study investigates a plug-flow structured anaerobic fixed-bed reactor (PF-AnFBR) for removal nine antibiotics representing different classes-an approach rarely explored in anaerobic systems. By integrating spatially resolved sampling along the reactor bed with advanced kinetic modeling, the study provides the first mechanistic evaluation of antibiotic mixture removal in a PF-AnFBR. COD removal remained high (COD > 97%) despite the presence of antibiotics, and significant removal was observed for trimethoprim (100%), sulfamethoxazole (83.3%), and enrofloxacin (81.3%). The first-order model accurately described COD removal, while the reversible biotransformation model (RevBio) successfully captured antibiotic fate (NRMSE < 3.5%), revealing class-specific mechanisms: fluoroquinolones dominated by adsorption (high K_D and k_sor), sulfonamides exhibiting reversible biotransformation, and trimethoprim characterized by highly irreversible biotransformation. Microbial community and KEGG-based functional analyses identified key taxa (e.g., Pseudomonas, Lactivibrio, Syntrophorhabdus, Methanothrix) and metabolic pathways (ABC transporters, cytochrome P450 enzymes) responsible for antibiotic transformation. By coupling reactor hydrodynamics, kinetic modeling, and microbial ecology, this study provides novel mechanistic insight into the removal of complex antibiotic mixtures in anaerobic fixed-bed reactors and supports PF-AnFBR as a robust technology for decentralized wastewater treatment.

Trans-anethole stands as a significant natural compound, finding extensive application as a flavoring ingredient across the food, cosmetics, perfumery, and pharmaceutical sectors. Trans-anethole also offers a variety of positive health benefits to humans, including anti-cancer, anti-inflammatory, anti-diabetic, immunomodulatory, and neuroprotective effects. In this study, we constructed a de novo trans-anethole pathway in E. coli by exploring the reduction of p-coumaric acid to p-coumaryl alcohol through carboxylate reductase and endogenous aldehyde reductase. First, an engineered Escherichia coli was created that could biosynthesise trans-anethole from glucose by the co-expression of aroG^fbr, tyrA^fbr,TAL, CAR, Sfp, ATF1, LtPPS1 and AIMT1. Second, by increasing the availability of SAM, we further increased the production of trans-anethole to 125 mg/L. Subsequently, the genes for transketolase I tktA and phosphoenolpyruvate synthase ppsA were further overexpressed to increase the availability of erythrose-4-phosphate and phosphoenolpyruvate, resulting in an increase in trans-anethole production to 223 mg/L. Finally, by knocking out the pheA gene to block the competitive pathway, the titer of trans-anethole was increased to 356 mg/L.

Biological pretreatment by white-rot fungi is a promising green technology for lignocellulose valorization, yet its industrial application is hampered by the high energy consumption of conventional steam sterilization. While a hurdle technology strategy combining low-temperature pasteurization and pH control has proven effective for wheat straw, its general applicability across diverse agricultural residues remains unclear. This study systematically compared the effects of low-temperature pasteurization (70 °C and 80 °C) with autoclaving (121 °C) on the pretreatment of wheat, rice, and rapeseed straw by Irpex lacteus at an initial pH of 4.5. The results revealed significant substrate-specific responses. For wheat and rapeseed straw, pasteurization achieved enzymatic saccharification yields comparable to or exceeding those of autoclaving, demonstrating excellent energy-saving potential. In contrast, rice straw required autoclaving at 121 °C to achieve maximum delignification (55.91% acid-insoluble lignin loss) and subsequent sugar release, likely due to its more recalcitrant native microbiota and structure. Pyrolysis-Gas Chromatography-Mass Spectrometry (Py-GC/MS) analysis confirmed that the S/G ratio decreased after pretreatment. Correlation analysis further established that the S/G ratio had a stronger negative correlation with glucose yield (r = - 0.94) than the total acid-insoluble lignin (AIL) content (r = - 0.90). These results suggest that the selective degradation of lignin structure, particularly the removal of S-units, is more closely associated with hydrolysis efficiency than total lignin removal. This work validates the viability of a low-temperature biological pretreatment strategy and underscores the necessity of tailoring protocols to specific feedstock characteristics to achieve efficient and economical biomass conversion.

2,3-Butanediol (2,3-BDO) is recognized for its industrial competitiveness and is predominantly produced in its enantiomerically pure form via microbial fermentation. The economical production of 2,3-BDO relies on the effective use of complex lignocellulosic materials and the advancement of inhibitor-resistant microbial strains. The robust expression of 2,3-BDO dehydrogenase in bacteria enhances volumetric productivity in inhibitor-rich lignocellulose hydrolysate. For instance, isolation of an inhibitor-resistant Klebsiella pneumoniae from palm oil effluent facilitated the synthesis of 75 g L⁻¹ of 2,3-BDO through separate hydrolysis and fermentation process. Conversely, the electrochemical detoxification of sugarcane bagasse hydrolysate increased the production of 2,3-BDO to 114.3 g L⁻¹ in Enterobacter aerogenes. Furthermore, deletion of glucose transporter (ptsG) in 2,3-BDO-producing bacteria mitigated carbon catabolite repression (CCR). Adaptive evolution of Paenibacillus polymyxa in non-detoxified wheat straw hydrolysate enhanced 2,3-BDO productivity to 0.72 g L⁻¹ h⁻¹. However, the rational engineering of yeast is complex, encompassing the heterologous expressions of xylose metabolism, 2,3-BDO dehydrogenase, and the deletion of the Crabtree effect. Nevertheless, partial disruption of the Crabtree effect in polyploid Saccharomyces cerevisiae resulted in increased production of 2,3-BDO (132 g L⁻¹) during the fed-batch fermentation of cassava hydrolysate. This review paper discusses the benefits and drawbacks of 2,3-BDO metabolism in both bacteria and yeast. The paper seeks to clarify the differences in 2,3-BDO production between detoxified and non-detoxified lignocellulosic hydrolysates. Further, the study illustrates the importance of generating 2,3-BDO from untreated lignocellulose via the development of microbial consortia. Economic factors that facilitate the commercialization of 2,3-BDO fermentation have been discussed in detail.

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.

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.

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.