Decellularization is an effective strategy to eliminate the immunogenicity of biomaterials to the host. The impact of raw material thickness on decellularization process was assessed. Three different thicknesses materials were selected and decellularized using an ultrasound-assisted method combined with tributyl phosphate. The results showed that Alaska pollock skin (0.23±0.06mm) was severely damage structurally after decellularization. Tilapia skin (0.64±0.12mm) exhibited higher decellularization efficiency and a relatively intact collagen fibre structure. Porcine skin (1.27±0.15mm), however, required additional decellularization treatment due to its dense structure. Based on these results, tilapia skin and porcine skin were selected for subsequent performance analysis. The results showed that the porosity (78.38%±4.06%) and swelling ratio (473.16%±15.62%) of tilapia skin were both higher than those of porcine skin. Hydroxyproline content and FTIR spectra confirmed preserved collagen triple-helix structure. Thermal stability increased after decellularization, with tilapia skin denaturation temperature reaching 56.2°C. Cytotoxicity testing revealed that decellularized tilapia skin extracts were non-cytotoxic, while porcine skin extracts showed mild cytotoxicity at higher concentrations. In conclusion, tilapia skin offers better decellularization efficiency, structural integrity, and biosafety under ultrasound-assisted treatment, making it a promising candidate for tissue engineering and wound repair.
A comparatively elevated concentration of nitrite in land-based intensive aquaculture wastewater typically suppresses the denitrification activity of bacteria within the wastewater, consequently leading to the nitrogen content in the effluent post-biological treatment surpassing the prescribed standard. This research concentrates on a strain of Rhodobacter azotoformans S7, which demonstrates high efficiency in nitrite denitrification, with the objective of addressing the aforementioned issue. Under optimal conditions (characterized by a weakly alkaline environment, a carbon-to-nitrogen ratio (C/N) of 15, an illumination intensity of 2300 Lux, a temperature of 30 °C, and 5% saturated dissolved oxygen), this strain exhibits a high nitrite removal efficiency. Based on these findings, γ-Fe₂O₃ magnetic nanoparticle-Pleurotus ostreatus composite carriers (MNPCs) were synthesized in this study to immobilize strain S7 for the continuous treatment of aquaculture wastewater. The results indicate that the MNPC-S7 system can operate stably for over 60 days under periodic illumination, achieving a high average nitrite removal efficiency of 98.91%. Mechanistic analysis reveals that the incorporation of γ-Fe₂O₃ not only optimizes the component ratio of polysaccharides (PS) and proteins (PN) in the biofilm (with a PN/PS ratio ranging from 2.5 to 4.0), but also accelerates the electron transfer rate and facilitates the enrichment of functional microorganisms, such as denitrifying bacteria. Overall, this bioaugmentation strategy offers an efficient and cost-effective technical solution for the treatment of aquaculture wastewater.
This study investigated the use of biochar as an adsorbent in an anoxic–oxic membrane bioreactor treating azo dye-containing wastewater. Three strategies were evaluated: (1) conventional reactor operation, (2) biochar addition to the mixed liquor, and (3) post-treatment using a fixed-bed biochar adsorption column. COD removal remained high (>98%) in strategies 1 and 2, indicating that biochar did not affect organic matter removal. Respirometric assays showed higher heterotrophic activity during the biochar-amended period, with the specific oxygen uptake rate increasing from 8.6 ± 0.7 to 10 ± 1.1mg O2 g-1 MLVSS h-1. Biochar incorporation improved dye removal (79.1% to 89.2%) and UVA254 reduction (38.1% to 45.5%). Batch assays showed that the dye removal rate increased from 0.64 to 1.79mg RBV-5R gVSS⁻¹ h⁻¹ during the biochar-amended period. Concurrently, membrane fouling was significantly mitigated, with an 87% reduction in fouling rate, accompanied by improved sludge filterability, as reflected by lower sCOD, CST, and SRF values. The highest effluent quality was achieved with post-treatment, resulting in 98.1% dye removal and 73.5% UVA254 reduction. Overall, in-reactor biochar improved dye removal, enhanced heterotrophic activity, and mitigated membrane fouling, whereas post- treatment provided additional polishing of residual dye, color, and aromatic compounds.
Cordyceps militaris (CMs), a valuable medicinal fungus parasitizing insect larva, is rich in bioactive adenosine (ADE) and cordycepin (COR). However, their poor stability and bioavailability limit practical applications. This study developed a scalable process for a double nanoemulsion (W1/O/W2) encapsulating CMs extract, subsequently converted into freeze-dried powder using a maltodextrin-trehalose carrier. Production was successfully scaled up to a 4kg batch. The system maintained a mean droplet size of 121.7 ± 5.7nm with acceptable polydispersity index and zeta potential values. Characterization via XRD, TGA and electron microscopy confirmed an amorphous structure and high thermal stability. Over 12 months, ADE and COR retention remained above 90% following first-order kinetics, with minor changes in moisture and dispersibility. Furthermore, the nanoformulation maintained the antioxidant activity of the CMs extract and stronger antibacterial activity against E. coli and S. aureus compared to the crude extract. This integrated workflow confirms the industrial potential of CMs double nanoemulsion powders for functional foods and nutraceutical applications.
β-poly(L-malic acid) (PMLA) is a biodegradable polyester of industrial interest owing to its promising applications in medicine and related fields. Producing PMLA using renewable feedstocks has emerged as a favorable strategy, among which Jerusalem artichoke tubers (JA) and molasses—abundant non-grain resources—are particularly attractive. This study innovatively developed a cost-effective poly(β-L-malic acid) (PMLA) production system by Aureobasidium melanogenum, integrating polyurethane matrix (PUM) immobilization and co-utilization of cane molasses and Jerusalem artichoke hydrolysate (JAH). Pristine 60 ppi PU was screened as the optimal carrier (277% immobilization capacity) for enhanced microbial adhesion and PMLA synthesis. Critically, JAH-molasses co-utilization completely replaced all basal medium components (carbon/nitrogen sources, inorganic salts), representing a 73% reduction of production cost. Repeated fed-batch fermentation in the PUM system achieved a maximum PMLA titer of 54.0g/L (0.75g/(L·h) productivity) with stable yields over 4 cycles. This integrated strategy pioneers a sustainable, scalable route for industrial PMLA production via low-cost agro-by-products and advanced immobilization.
Natural deep eutectic solvents (NADES) are emerging green extraction systems offering a sustainable alternative to conventional chemical solvents. Despite their popularity, the integration of NADES into downstream processing still remains largely unexplored. The present study investigated the dual functionality of NADES for both sustainable extraction of the bioactive fraction as well as its direct utilization as a functional medium component. A betaine:glycerol-based NADES (Bet:Gly2) was synthesized and used for the extraction of bioactive fraction from grape juice waste. The NADES-extracted fraction showed significantly higher bioactive content, including polyphenols and dietary fibre, compared with the conventional hydroethanolic solvent. The same bioactive fraction (PDBF) was used directly to develop a novel food-grade NADES-based functional medium. Optimization of medium components was carried out using RSM-BBD, and it was found that inclusion of 9.77% PDBF, 3.91g/L Yeast extract, 1.89g/L L-Glutamine, and 1.22g/L L-Proline were optimal for probiotic growth. Further, the ability of the functional medium to support probiotic growth and metabolic activities was confirmed by cell counts and short-chain fatty acid (SCFA) production (postbiotics). Safety evaluation of the food-grade NADES-based functional medium on the mammalian cell line HEK293 showed no cytotoxicity, thereby indicating its in vitro cytocompatibility.
Biogenic coalbed methane (CBM) is generated through microbial degradation of coal organic matter, yet the synergistic effects of biotic and abiotic factors across coal ranks remain unclear. This study presents parallel comparative experiments on raw coal, sterile culture medium immersion (abiotic), and microbial degradation (biotic) to reveal their differential modification characteristics. Abiotic immersion primarily alters coal composition and pore structure through physical swelling and extraction, without significant organic matter decomposition. In contrast, microbial degradation exhibits strong coal-rank dependence. For low-rank coal, biological consumption dominates, reducing organic matter content, increasing aromatization, and enhancing pore development. For middle-rank coal, a competitive balance emerges between microbial degradation and solubilization, leading to net organic matter changes and complex pore responses. For high-rank coal, biosolubilization becomes dominant, manifesting as apparent organic matter increase, limited oxidation, and formation of micropores. Based on these findings, a rank-dependent coupled evolution model of the “water–microbe–coal” system is proposed. This model clarifies the synergistic contributions of abiotic and biotic processes across different coal ranks, deepens the mechanistic understanding of biogenic CBM generation, and provides a theoretical foundation for resource assessment and enhanced development of biogenic CBM as a renewable unconventional natural gas resource.
Transaminase-based amino acid synthesis is often limited by unfavorable reaction equilibria. Here, we show that the poorly soluble amino acid D-tyrosine can be synthesized from 4-hydroxyphenylpyruvic acid (4-HPP) to near completion by D-amino acid transaminase (DAAT) in a whole-cell system using equimolar D-alanine, without externally added equilibrium-shifting enzymes. Our approach relies on dual equilibrium displacement driven by in situ product precipitation and metabolic coproduct removal. Under homogeneous conditions with purified DAAT, the equilibrium constant for the reaction between 4-HPP and D-alanine was determined to be 0.59, indicating that the intrinsic equilibrium disfavors D-tyrosine formation. At high substrate loadings, however, conversion greatly exceeded the equilibrium limit observed under homogeneous conditions and closely matched theoretical predictions based on precipitation-driven clamping of dissolved D-tyrosine at its solubility limit. Replacing the purified enzyme with a whole-cell biocatalyst further increased conversion through a metabolic pyruvate sink, ultimately enabling 98% conversion of 50mM 4-HPP with equimolar D-alanine. The synthetic utility of this strategy was demonstrated by preparative-scale stereoinversion of L-tyrosine using L-amino acid deaminase and DAAT, affording D-tyrosine in >99% ee and 36% isolated yield. These results provide a quantitative framework for applying dual equilibrium displacement to the synthesis of other poorly soluble amino acids.
The antimicrobial peptide LL-37, the only cathelicidin present in the human body, represents a central component of human innate immunity. Its broader application is unfortunately limited due to high production costs, purification challenges, and peptide instability. This study builds on the already established, scalable, plant-based production of LL-37 in barley seeds, where it is expressed in a fusion with maltose-binding protein and an endoplasmic reticulum retention signal. The fusion protein MBP_rhLL-37 was extracted from lyophilized immature barley grains and enriched by ammonium sulphate precipitation. Proteolytic processing of the fusion protein was evaluated using the epidermal serine proteases KLK5, KLK7, KLK8, and KLK14, and the antimicrobial activity of the resulting products was assessed against Escherichia coli TOP10. Among the tested proteases, KLK14 showed the most efficient cleavage of the fusion protein and generated LL-37-immunoreactive products associated with antimicrobial activity. In addition, the barley extract containing MBP_rhLL-37 was successfully incorporated into a cream formulation suitable for topical delivery studies. Overall, this work demonstrates that barley-based molecular farming, combined with non-chromatographic purification and physiologically relevant protease activation, provides a practical, cost-effective, and environmentally sustainable platform for the LL-37 application in topical formulation.
Aminated supports are utilized in the preparation of immobilized antibodies to be used as biosensors. However, these supports have an anionic exchanger feature that can promote some false positives and thus affect the detection limit. Serum albumin is a protein able to interact with multiple moieties and proteins. This paper attempts to study how the surface density of amino groups in the support surface or the coating of highly activated supports with dextran can eliminate the unspecific adsorption of proteins. Using supports with 11 micromols of ethylenediamine per g of support (10% of the maximum activation degree) and after only 1 h of incubation of the support in serum, no proteins were adsorbed on the support. However, after 24 h of incubation some protein adsorption was detected. The sequential blocking of the highly activated support with dextran-Asp (40%)-aldehyde(60%) in a sequential modification with dextran of 100, 25 and 6 KD fully prevented the adsorption of albumin from serum after 1 h of incubation. However, some protein adsorption was observed after 24 h. That way, the full prevention of the albumin on aminated supports cannot be fully prevented, but the rate of adsorption can be significantly decreased designing an adequate support.
N‑Acetylneuraminic acid (Neu5Ac) has demonstrated a variety of physiological benefits, including antiviral and antioxidant activities, as well as immune‑regulatory functions. Owing to its considerable demand in both basic research and commercial applications, such as pharmaceuticals, cosmetics, and food, the establishment of a green, efficient, and low‑cost biosynthetic route for Neu5Ac is of major significance. In this work, we designed an Escherichia coli platform for efficient Neu5Ac biosynthesis by constructing and optimizing multienzyme complexes mediated by a specific self‑assembling peptide pair. The resulting RIDD‑RIAD‑based assemblies improve the catalytic efficiency of the sequential pathway cascade, thus enhancing metabolic flux and product titer. In addition, the endogenous biosynthetic pathway for the key precursor UDP‑GlcNAc was strengthened, leading to a remarkable boost in Neu5Ac production. Through scaled‑up fermentation in a 5‑L bioreactor, the strain achieved a high Neu5Ac titer of 74.116g/L and a productivity of 1.370g/L/h. The pronounced improvement in Neu5Ac bio-production highlights the substantial potential of the construction of synthetic multienzyme complexes as a practical and effective strategy for spatially organizing metabolic pathways, offering a model for advanced chassis design in synthetic biology.
Bacillus clausii F7 is an indigenous thermophilic bacterium isolated from an oil reservoir that produces lipopeptide biosurfactants with antimicrobial activity. However, compared with other Bacillus species, strategies to enhance biosurfactant production and improve the operational stability of B. clausii remain largely unexplored. This study optimized calcium alginate bead formation, compared biosurfactant production kinetics between free and immobilized cells, evaluated bead stability, and characterized the produced biosurfactants. Biosurfactants were produced in Stone Mineral Salt Solution (SMSS) medium, and bead formation was optimized using Response Surface Methodology (RSM) based on a Central Composite Design (CCD). The optimum formulation consisted of an alginate volume of 9.77mL, alginate pH of 7.06, and a bead diameter of 5.3mm. Immobilized cells reached maximum biosurfactant production after 48h and produced a higher maximum yield (0.206g/L) than free cells (0.156g/L). The optimized beads remained stable through six consecutive production cycles, yielding a cumulative biosurfactant concentration of 0.933g/L, while maintaining cell viability during 28 days of storage at 4 °C. Thin-layer chromatography and UHPLC–MS/MS confirmed lipopeptide biosurfactants, including surfactin homologues (m/z 1021.69 and 1022.3) and iturin-like compounds (m/z 1106 and 1125). Overall, the optimized immobilized-cell system enhanced biosurfactant production, operational stability, and reusability.
This study investigates a microwave-assisted extraction (MAE) process for the recovery of astaxanthin-rich extract (ARE) from Paracoccus carotinifaciens using ethyl acetate as a recyclable green solvent, integrating mechanistic modeling and techno-economic evaluation. Experimental data combined with computational fluid dynamics (CFD) revealed that microwave-induced thermal gradients and diffusion-driven mass transfer govern pigment release and recovery. Operating conditions (350–400 W; solid–liquid ratio ∼1:30) yielded approximately 40 mg AXT eq./g within 40 min, while preventing thermal degradation. The kinetic model showed strong agreement with experimental data (R² = 0.98). The recovered ARE was successfully incorporated into a lip balm formulation, exhibiting uniform reddish–orange coloration and good stability. A preliminary techno-economic assessment indicated strong economic potential, with a return on investment (ROI) of 168% and a net present value (NPV) of $648.8 million, supporting the potential industrial scalability and economic feasibility of the proposed process. Overall, this work provides a framework combining process optimization, mechanistic understanding, and application potential for the sustainable recovery of microbial carotenoids.
The industrial application of certain GH11 xylanases, including XynASP from A. saccharolyticus JOP 1030–1, is constrained by inadequate thermostability. Here, we engineered disulfide bonds at the linker-linker interface of XynASP. A single mutant S134C and a disulfide-bonded mutant S134C/N178C with improved thermostability or catalytic activity were successfully screened. Enzymatic characterization showed that mutant S134C increased the half-life at 50 °C by 3.6‑fold and exhibited a substantial 9.5 °C increase in melting temperature (Tm), though it decreased specific activity. In contrast, the disulfide-bonded mutant S134C/N178C exhibited a 4.5-fold increase in specific activity and a moderate 1.6-fold improvement in thermostability, with the Tm values remaining comparable to the wild-type XynASP. Structural analysis and molecular dynamics simulations revealed that S134C enhanced local rigidity of the linker region through formation of a hydrophobic cluster, whereas the disulfide bond between Cys134 and Cys178 in the double mutant created a "rigid‑yet‑tunable" molecular scaffold, which optimized the microenvironment of the substrate-binding pocket and the dynamic correlation network, leading to a 5.4‑fold increase in catalytic efficiency (kcat/Km). This study establishes linker-linker interface disulfide bonds as a regulator of GH11 xylanase catalysis, revealing a new strategy for mitigating the stability-activity trade-off.
