Bioprocess and Biosystems Engineering最新文献

In light of the growing demand for novel biocatalysts and enzyme production methods, this study aimed to evaluate the potential of Aspergillus tubingensis for producing lipase under submerged culture investigating the influence of culture time and inducer treatment. Moreover, this study also investigated conditions for the immobilization of A. tubingensis lipase by physical adsorption on styrene-divinylbenzene beads (Diaion HP-20), for these conditions to be applied to an alternative immobilization system with a packed-bed reactor. Furthermore, A. tubingensis lipase and its immobilized derivative were characterized in terms of their optimal ranges of pH and temperature. A. tubingensis was shown to be a good producer of lipase, obviating the need for inducer addition. The enzyme extract had a hydrolytic activity of 23 U mL^-1 and achieved better performance in the pH range of 7.5 to 9.0 and in the temperature range of 20 to 50 °C. The proposed immobilization system was effective, yielding an immobilized derivative with enhanced hydrolytic activity (35 U g^-1), optimum activity over a broader pH range (5.6 to 8.4), and increased tolerance to high temperatures (40 to 60 ℃). This research represents a first step toward lipase production from A. tubingensis under a submerged culture and the development of an alternative immobilization system with a packed-bed reactor. The proposed system holds promise for saving time and resources in future industrial applications.

Ginsenoside compound K (CK) holds significant potential for application in the pharmaceutical industry, which exhibits numerous pharmacological activity such as cardioprotective and antidiabetic. However, the difficult separation technique and limited yield of CK hinder its widespread use. The study investigated the process of converting ginsenoside CK using β-glucosidase. It aimed to determine the specific site where the enzyme binds and the most favorable arrangement of the enzyme. Molecular docking was also employed to determine the interaction between β-glucosidase and ginsenosides, indicating a strong and spontaneous contact force between them. The effectiveness of the conversion process was further improved using a "green" deep eutectic solvent (DES). A univariate experimental design was used to determine the composition of DES and the optimal hydrolysis conditions for β-glucosidase to convert ginsenoside Rb1 into ginsenoside CK. The employment of β-glucosidase enzymatic hydrolysis in the synthesis of rare ginsenoside CK applying the environmentally friendly solvent DES is not only viable and effective but also appropriate for industrial use. The characterization methods confirmed that DES did not disrupt the structure and conformation of β-glucosidase. In ChCl:EG = 2:1 (30%, v/v), pH 5.0 of DES buffer, reaction temperature 50 ℃, enzyme substrate mass ratio 1:1, after 36 h of reaction, the CK yield was 1.24 times that in acetate buffer, which can reach 86.2%. In this study, the process of using β-glucosidase enzymatic hydrolysis and producing rare ginsenoside CK in green solvent DES is feasible, efficient and suitable for industrial production and application.

Erythritol is a natural non-caloric sweetener, which is produced by fermentation and extensively applied in food, medicine and chemical industries. The final step of the erythritol synthesis pathway is involved in erythritol reductase, whose activity and NADPH-dependent become the limiting node of erythritol production efficiency. Herein, we implemented a strategy combining molecular docking and thermal stability screening to construct an ER mutant library. And we successfully obtained a double mutant ER^K26N/V295M (ER*) whose catalytic activity was 1.48 times that of wild-type ER. Through structural analysis and MD analysis, we found that the catalytic pocket and the enzyme stability of ER* were both improved. We overexpressed ER* in the engineered strain ΔKU70 to obtain the strain YLE-1. YLE-1 can produce 39.47 g/L of erythritol within 144 h, representing a 35% increase compared to the unmodified strain, and a 10% increase compared to the strain overexpressing wild-type ER. Considering the essentiality of NADPH supply, we further co-expressed ER* with two genes from the oxidative phase of PPP, ZWF1 and GND1. This resulted in the construction of YLE-3, which exhibited a significant increase in production, producing 47.85 g/L of erythritol within 144 h, representing a 63.90% increase compared to the original chassis strain. The productivity and the yield of the engineered strain YLE-3 were 0.33 g/L/h and 0.48 g/g glycerol, respectively. This work provided an ER mutation with excellent performance, and also proved the importance of cofactors in the process of erythritol synthesis, which will promote the industrial production of erythritol by metabolic engineering of Y. lipolytica.

This study investigated cellobionate production from a lignocellulosic substrate using Neurospora crassa HL10. Utilizing NaOH-pretreated wheat straw as the substrate obviated the need for an exogenous redox mediator addition, as lignin contained in the pretreated wheat served as a natural mediator. The low laccase production by N. crassa HL10 on pretreated wheat straw caused slow cellobionate production, and exogenous laccase addition accelerated the process. Cycloheximide induced substantial laccase production in N. crassa HL10, enabling the strain to yield approximately 57 mM cellobionate from pretreated wheat straw (equivalent to 20 g/L cellulose), shortening the conversion time from 8 to 6 days. About 92% of the cellulose contained in the pretreated wheat straw is converted to cellobionate. In contrast to existing methods requiring pure cellobiose or cellulase enzymes, this process efficiently converts a low-cost feedstock into cellobionate at a high yield without enzyme or redox mediator supplementation.

Polyhydroxyalkanoate (PHA) is an environmental alternative to petroleum-based plastics because of its biodegradability. The polymer properties of PHA have been improved by the incorporation of different monomers. Traditionally, the monomer composition of PHA has been analyzed using gas chromatography (GC) and nuclear magnetic resonance (NMR), providing accurate monomer composition. However, sequential analysis of the thermal properties of PHA using differential scanning calorimetry (DSC) remains necessary, providing crucial insights into its thermal characteristics. To shorten the monomer composition and thermal property analysis, we directly applied DSC to the analysis of the obtained PHA film and observed a high correlation (r² = 0.98) between melting enthalpy and the 3-hydroxyhexanoate (3-HHx) mole fraction in the polymer. A higher 3-HHx fraction resulted in a lower melting enthalpy as 3-HHx provided the polymer with higher flexibility. Based on this, we selected the poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (P(3HB-co-3HHx)) producing strain from Cupriavidus strains that newly screened and transformed with vectors containing P(3HB-co-3HHx) biosynthetic genes, achieving an average error rate below 1.8% between GC and DSC results. Cupriavidus sp. BK2 showed a high 3-HHx mole fraction, up to 10.38 mol%, with T_m (℃) = 171.5 and ΔH of T_m (J/g) = 48.0, simultaneously detected via DSC. This study is an example of the expansion of DSC for PHA analysis from polymer science to microbial engineering.

The synthesis of magnesium hydroxide nanoparticles (Mg(OH)₂ NPs) using plant extracts are known to be a practical, economical, and an environmentally friendly approach. In this work, Mg(OH)₂ NPs were synthesized using aqueous leaf extract of Tinospora cordifolia, a medicinal plant commonly found in India. The synthesized Mg(OH)₂ NPs were characterized using various spectroscopic techniques. The ultraviolet–visible (UV–Vis) absorption peak of the Mg(OH)₂ NPs was detected at 289 nm, Fourier transform infrared (FTIR) analysis confirmed the presence of various functional groups, and X-ray diffraction (XRD) patterns revealed the well-crystallized structure of the Mg(OH)₂ NPs. High-resolution transmission electron microscopy (HR-TEM) and scanning electron microscopy (SEM) analyses depicted spherical morphology and an average particle size (PS) of 27.71 nm. The energy-dispersive X-ray (EDX) analysis confirmed the presence of C, O, and Mg elements, and the X-ray photoelectron spectroscopy (XPS) survey spectrum confirmed the elements for the Su 1 s peak at 280.2 eV. The dynamic light scattering (DLS) analysis displayed an average PS of 54.3 nm, and the Zeta potential (ZP) was of 9.89 mV. The fabricated Mg(OH)₂ NPs displayed notable antibacterial activity against S. epidermidis, E. coli, and S. aureus. In addition, these NPs exhibited strong antioxidant properties (> 75%) based on DPPH, ABTS, and hydrogen peroxide (H₂O₂) assays. Further, the same NPs exerted a potent anti-inflammatory activity (> 65%) based on COX-1 and COX-2 evaluations. The anti-Alzheimer’ disease (AD) potential of Mg(OH)₂ NPs was assessed through effective inhibition (> 70%) of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activities. Molecular docking (MD) studies confirmed that caryophyllene has higher binding affinity with AChE (−5.3 kcal/mol) and BuChE (−6.4 kcal/mol) enzymes. This study emphasizes the green synthesis of Mg(OH)₂ NPs using T. cordifolia as a plant source and highlights their potential for biomedical applications.

The probiotic fermentation of the bioactive substance gamma-aminobutyric acid (GABA) is an attractive research topic. There is still room for further improvement in reported GABA fermentation methods based on a single substrate (l-glutamic acid or l-monosodium glutamate). Here, we devised a pH auto-buffering strategy to facilitate the fermentation of GABA by Levilactobacillus brevis CD0817. This strategy features a mixture of neutral monosodium l-glutamate plus acidic l-glutamic acid as the substrate. This mixture provides a mild initial pH; moreover, the newly dissolved l-glutamic acid automatically offsets the pH increase caused by substrate decarboxylation, maintaining the acidity essential for GABA fermentation. In this study, a flask trial was first performed to optimize the GABA fermentation parameters of Levilactobacillus brevis CD0817. The optimized parameters were further validated in a 10 L fermenter. The flask trial results revealed that the appropriate fermentation medium was composed of powdery l-glutamic acid (750 g/L), monosodium l-glutamate (34 g/L [0.2 mol/L]), glucose (5 g/L), yeast extract (35 g/L), MnSO₄·H₂O (50 mg/L [0.3 mmol/L]), and Tween 80 (1.0 g/L). The appropriate fermentation temperature was 30 °C. The fermenter trial results revealed that GABA was slowly synthesized from 0–4 h, rapidly synthesized until 32 h, and finally reached 353.1 ± 8.3 g/L at 48 h, with the pH increasing from the initial value of 4.56 to the ultimate value of 6.10. The proposed pH auto-buffering strategy may be popular for other GABA fermentations.

The production of keratinases was evaluated in submerged fermentation with Aspergillus niger and by pigs’ swine hair in a batch bioreactor. Experimental planning was performed to assess the interaction between different variables. The enzyme extract produced was characterized at various pH and temperatures and subjected to enzyme concentration using a biphasic aqueous system and salt/solvent precipitation techniques. In addition, the substrate’s potential in reducing hexavalent chromium from synthetic potassium dichromate effluent with an initial concentration of 20 mg L⁻¹ of chromium was evaluated. The resulting enzyme extract showed 89 ± 2 U mL⁻¹ of keratinase. The enzyme concentration resulted in a purification factor of 1.3, while sodium chloride/acetone and ammonium sulfate/acetone resulted in a purification factor of 1.9 and 1.4, respectively. Still using the residual substrate of swine hair from the fermentation, a 94% reduction of hexavalent chromium concentration occurred after 9 h of reaction. Thus, the study proved relevant for producing keratinases, with further environmental applicability and the possibility of concentrating the extract via low-cost processes.

Perfusion cell-culture mode has caught industrial interest in the field of biomanufacturing in recent years. Thanks to new technology, perfusion-culture processes can support higher cell densities, higher productivities and longer process times. However, due to the inherent operational complexity and high running costs, the development and design of perfusion-culture processes remain challenging. Here, we present a model-based approach to design optimized perfusion cultures of Chinese Hamster Ovary cells. Initially, four batches of bench-top reactor continuous-perfusion-culture data were used to fit the model parameters. Then, we proposed the model-based process design approach, aiming to quickly find out the “theoretically optimal” operational parameters combinations (perfusion rate and the proportion of feed medium in perfusion medium) which could achieve the target steady-state VCD while minimizing both medium cost and perfusion rate during steady state. Meanwhile, we proposed a model-based dynamic operational parameters-adjustment strategy to address the issue of cell-growth inhibition due to the high osmolality of concentrated perfusion medium. In addition, we employed a dynamic feedback control method to aid this strategy in preventing potential nutrient depletion scenarios. Finally, we test the feasibility of the model-based process design approach in both shake flask semi-perfusion culture (targeted at 5 × 10⁷ cells/ml) and bench-top reactor continuous perfusion culture (targeted at 1.1 × 10⁸ cells/ml). This approach significantly reduces the number of experiments needed for process design and development, thereby accelerating the advancement of perfusion-mode cell-culture processes.

Antibiotics are widely used as fungicides because of their antibacterial and bactericidal effects. However, it is necessary to control their dosage. If the amount of antbiotics is too much, it cannot be completely metabolized and absorbed, will pollute the environment, and have a great impact on human health. Many antibiotics usually left in factory or aquaculture wastewater pollute the environment, so it is vital to detect the content of antibiotics in wastewater. This article summarizes several common methods of antibiotic detection and pretreatment steps. The detection methods of antibiotics in wastewater mainly include immunoassay, instrumental analysis method, and sensor. Studies have shown that immunoassay can detect deficient concentrations of antibiotics, but it is affected by external factors leading to errors. The detection speed of the instrumental analysis method is fast, but the repeatability is poor, the price is high, and the operation is complicated. The sensor is a method that is currently increasingly studied, including electrochemical sensors, optical sensors, biosensors, photoelectrochemical sensors, and surface plasmon resonance sensors. It has the advantages of fast detection speed, high accuracy, and strong sensitivity. However, the reproducibility and stability of the sensor are poor. At present, there is no method that can comprehensively integrate the advantages. This paper aims to review the enrichment and detection methods of antibiotics in wastewater from 2020 to the present. It also aims to provide some ideas for future research directions in this field.