Bioprocess and Biosystems Engineering最新文献

Dark fermentation represents a sustainable and promising approach for biohydrogen generation. However, achieving high yields depends on understanding the complex microbial interactions driving the process. This study used genome-centric metagenomics to analyze microbial communities from 11 hydrogen-producing reactors. In total, 44 metagenome-assembled genomes (MAGs) were analyzed in detail. High-yield reactors demonstrated a strong synergy between hydrogen-producing bacteria (HPB) and lactic acid bacteria (LAB), particularly Clostridium butyricum and Clostridium beijerinckii. These species encode the electron-transferring flavoprotein-lactate dehydrogenase complex (EtfAB-ldh complex), enabling hydrogen production from lactic acid. In contrast, reactors with lower hydrogen yields exhibited a higher prevalence of hydrogenotrophic microorganisms, including homoacetogens and methanogens, which redirected electron flow toward competing pathways, thereby decreasing hydrogen output. These results emphasize the importance of promoting HPB while suppressing hydrogen consumers to maintain an optimal microbial community. By linking community composition with metabolic potential, this study provides a framework for improving reactor performance, increasing hydrogen yields, and advancing sustainable hydrogen production from organic waste streams.

The Catharanthus roseus herbs are the source of pharmaceutical alkaloids, including the antihypertensive ajmalicine and antineoplastics vincristine and vinblastine, whose global shortages have recently compromised medical therapies. Suspension culture represents a sustainable alternative for production but requires well-characterized cell lines for efficient bioprocess development. In this study, a suspension culture system of the novel CRBY-1 (Catharanthus roseus Bright Yellow-1) cell line has been established and comprehensively characterized. Cell proliferation and morphology analyses showed predominantly homogeneous oval cells (~ 108 μm) forming small aggregates of 1-4 cells, achieving a maximum dry biomass of 10 g/L. Growth kinetics revealed a specific growth rate of 0.49 d⁻¹, doubling time of 1.3-1.4 d, and a cell respiration rate of 0.0066 mg O₂ g⁻¹ s⁻¹. Nutrient uptake profiling indicated that phosphate and oxygen, rather than carbon, became limiting during mid-culture, influencing biomass formation and nitrogen assimilation. Alkaloid production analysis showed that CRBY-1 synthesized both ajmalicine and catharanthine at notable levels. For ajmalicine, the cell line produced 55 mg per kg of biomass inside the cells and 13 mg per litter in the culture medium. For catharanthine, production reached 133 mg per kg of biomass intracellularly and 13 mg per litter extracellularly. These values exceed those previously reported for callus cultures and plant tissues. Vincristine and vinblastine, however, were not detected in the suspension cultures. The cell line exhibited growth-associated alkaloid production, stable morphology, and high metabolic activity, reinforcing its potential for future scale-up in bioreactor systems. These findings provide critical insights for the future valorisation of CRBY-1 in sustainable, scalable production of high-value alkaloids and or other bioactive molecules.

Nattokinase is a high-valued functional component produced during natto fermentation, and both its activity level and production yield are of significant importance for industrial applications. This study aimed to optimize and scale up the fermentation process for nattokinase production. We first optimized the liquid fermentation process for nattokinase through single-factor and response surface methodology (RSM) experiments, determining the optimal medium composition. Under the optimized conditions, a nattokinase activity of 4257 IU/mL was attained in shake-flask fermentation. Subsequently, a fed-batch cultivation process was employed in pilot-scale experiments. In a 20 L fermenter, dynamic control of oxygen supply and optimization of feed addition timing were implemented, resulting in an increase of the nattokinase activity to 23,074 IU/mL. When scaling up to a 200 L fermenter, an innovative strategy was adopted to overcome the oxygen transfer efficiency bottleneck. This approach involved low-speed startup, stirring-dominated oxygen control, and strict ventilation restrictions. As a result, the nattokinase activity reached 21,312 IU/mL, equivalent to 92.36% of the activity achieved in the 20 L fermenter. This optimized and scalable process provides a stable and controllable technical solution for pilot-scale upscaling, and establishes a strong foundation for large-scale industrial production of nattokinase.

Upstream bioprocessing is a very complex system and requires rapid responses to process deviations. Mammalian cell culture processes are conventionally monitored for process-related and cell growth-related parameters, including pH, dissolved oxygen, viable cell density, cell viability, and key analyte concentrations that serve as primary indicators of the metabolic state of the cell culture. Raman spectroscopy (RS) has been increasingly applied as a viable inline process analytical technology (PAT) tool for cell culture monitoring and prediction of key analytes and attributes. The primary limitation to RS in these measurements is fluorescence (also referred to as sample-induced fluorescence), which interferes with the Raman signal and creates noise that makes detection of the signal from the analytes difficult. As a result, fluorescence interference decreases the signal to noise ratio (SNR) of the acquired spectra and increases the limit of detection (LOD) of analytical methods. Time-gated Raman spectroscopy (TGRS) takes advantage of the temporal delay between inelastic light scatter (Raman signal) and fluorescence emission to reduce interference from fluorescence. In this study, a pure component modeling approach and Net Analyte Signal (NAS) were applied to calculate the SNR and LOD of independent CHO cell culture samples. By reducing fluorescence interference, improving the SNR and LOD, TGRS enhanced the detectability of five key analytes in the cell culture samples, facilitating accurate monitoring and detection of analytes in a complex bioprocess system, thereby demonstrating its viability as a PAT tool for upstream bioprocess environment.

In this study, we developed a reusable low-adhesion metallic cell culture surface having microscale structures using nanosecond pulsed laser processing. Titanium alloy disks were mirror-polished and laser-processed to create microstructures with a pitch of 15 μm, smaller than typical cell size. The cytocompatibility of the developed surfaces was confirmed, showing comparable viability to standard plastic dishes. On the other hand, the cells on the laser-processed surfaces exhibited suppressed lamellipodia formation and maintained a rounded morphology and the area of adhered cells was significantly inhibited compared to polished surfaces, indicating reduced adhesion. Further, by applying PBS jet flow to the culture surface, it has been demonstrated that the cells on the micro-structured surfaces formed significantly larger detachment zones under PBS jet flow, confirming weakened adhesion strength. Furthermore, intact cell sheets could be detached from the laser-processed surfaces by pipetting, whereas cells on polished surfaces remained adherent. These results suggest that the developed culture surface enables on-demand cell detachment through physical stimuli without enzymatic treatment, maintaining cell-cell junctions and extracellular matrix integrity. This technology offers potential for applications in cell sheet engineering and enzyme-free cell harvesting, contributing to cost-effective and sustainable cell-based applications. Future work should investigate cell proliferation and migration behavior to further validate its utility for industrial tissue engineering platforms.

Phenolic compounds in oats contribute to their health benefits but predominantly exist in insoluble bound forms with low bioaccessibility. To address this issue, this study developed a phased processing strategy combining enzymatic hydrolysis and Monascus fermentation to enhance the release of bioactive phenolic in oats. Results showed that adding cellulase in the mid-fermentation stage effectively increased the phenolic content by 21.23 times (23.34 mg GAE/g DW), compared with unfermented oats. HPLC analysis revealed substantial increases in free phenolic acids, with vanillic acid and chlorogenic acid contents rising to 215.22 mg/kg (35.92-fold) and 150.90 mg/kg (16.00-fold), respectively. Structural analysis via scanning electron microscopy confirmed the degradation of oat cell walls, supporting microbial growth and facilitating phenolic compound release. The free phenolic fractions exhibited potent antioxidant activities, which were strongly correlated (r > 0.91, p ≤ 0.001) with chlorogenic acid, quercetin, and vanillic acid content. These results demonstrated that the combined microbial-enzymatic approach was a highly effective bioprocessing strategy for producing value-added oat products with enhanced phenolic bioaccessibility and antioxidant capacity.

Biohydrogen (BioH₂) production from waste resources, such as food waste, is a potential source of sustainable and clean energy. Previous literature has reported enhancement in the kinetics and yield of dark fermentation for bioH₂ production using sonication. However, the mechanism by which sonication affects the cellular metabolism has remained largely unexplored. The present study aims to investigate the effect of ultrasound on the metabolic network of Clostridium pasteurianum during the dark fermentation of food waste hydrolysate and to elucidate the underlying mechanism using metabolic flux analysis (MFA). A metabolic flux model was developed to determine the impact of sonication on intracellular metabolite fluxes. Hexose sugar uptake increased by ~ 47% with sonication, while butyrate and acetate fluxes at the acetyl-CoA node increased by ∼9% and ∼94%, respectively. Sonication improved bioH₂ yield by ∼22%, and the acetate-to-butyrate (A/B) ratio by ∼37%. These results pointed out that bioH₂ production is linked to carbon flux at the acetyl-CoA node. A higher flux towards the acetate route (compared to the butyrate route) enhances hydrogen yield. Based on these results, a hypothetical MFA analysis (with sonication) was conducted for two cases: (1) complete redirection of carbon flux at the acetyl-CoA node to the acetate route, and (2) doubling the uptake flux of hexose sugars. For the first case, bioH₂ enhanced from 4.13 to 6.47 mmol/L⋅h, while in second case, bioH₂ flux of 14.53 mmol/L⋅h was predicted by MFA model. These results could be useful for the genetic engineering of microbial strains for enhanced bioH₂ production.

Volatile fatty acids (VFAs) derived from organic waste offer promising and cost-effective carbon sources for the production of microbial lipids. This study demonstrates the significant influence of nitrogen nutrition on cell proliferation and microbial lipid synthesis in Yarrowia lipolytica during high-concentration acid cultivation. Further investigations into nitrogen sources revealed that NH₄Cl and urea are suitable options for cultivating Y. lipolytica to produce microbial lipids, resulting in lipid yields ranging from 2.00 to 2.50 g/L. Moreover, pH fluctuations were found to be influenced by both the nitrogen source and acid utilisation, with pH adaptation helping alleviate acid inhibition caused by high-concentration VFAs. Under optimised cultivation conditions, the highest yield of microbial lipids reached 4.00 g/L, accompanied by a dry cell weight of 9.91 g/L and a microbial lipid content of 40.37%, consisting predominantly of C16 ~ 18 fatty acids. These findings highlight the central role of nitrogen metabolism and pH adaptation in enhancing VFA assimilation, offering guidance for cost-effective microbial lipid production from organic waste streams.