Polyhydroxyalkanoates (PHAs) are promising bio-based and biodegradable polymers that can contribute to the transition towards a circular bioeconomy, particularly when produced from renewable feedstocks. Nevertheless, the use of mixed microbial cultures (MMC) and real agri-food residues poses challenges related to substrate variability, process stability and control of polymer composition. In this study, a three-stage process was investigated to produce poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) from agri-food residues. The process consisted of: (i) acidogenic fermentation of cattle slurry and corn residues to generate a carboxylic acid (CA)-rich stream; (ii) microbial selection under feast-famine conditions to enrich PHA-accumulating microorganisms; and (iii) PHA accumulation in a fed-batch reactor. Two operational setups, differing in the pretreatment type, were compared to evaluate possible effects on PHA production: setup 1 used centrifuged fermentate, whereas setup 2 included an additional 0.22-µm filtration step. Acidogenic fermentation produced up to 18.6gCOD/L of CAs, mainly acetic, butyric and propionic acids, with production strongly influenced by substrate ratio, hydraulic retention time and pH. The CA-rich fermented liquid was used to select MMC enriched with PHBV-accumulating microorganisms and to tune the PHBV composition during the subsequent accumulation phase. In setup 1, storage yield (mean 0.33 ± 0.23 gCODPHA/gCODCA), PHA titre (mean 0.81 ± 0.33g/L) and intracellular PHA contents (4.0-21.0% w/w of cell dry weight) remained relatively low, likely due to the use of unfiltered fermentation effluent. In setup 2, the introduction of a filtration step and improved microbial selection led to higher and more stable conversion of CAs into PHAs, reaching mean storage yields of 0.78 ± 0.27 gCODPHA/gCODCA, mean PHA titre of 1.70 ± 0.44g/L and intracellular PHA values up to 57.0% w/w. The hydroxyvalerate (HV) monomer content showed temporal variability, ranging from 14.0-32.0% w/w in setup 1 and from 13.0-35.0% w/w in setup 2, with values generally within or close to the target range required to improve polymer flexibility and processability. Microbial community analysis revealed a marked reduction in genus richness following feedstock filtration and the progressive dominance of specialised PHA-storing genera, indicating effective selection pressure under feast-famine operation and supporting the observed improvements in polymer accumulation. Overall, the results indicate that MMC-based PHBV production from agri-food residues can achieve polymer contents comparable to those reported for similar systems using complex substrates, while highlighting the importance of feedstock pretreatment and process control. The study provides insights into the integration of acidogenic fermentation with MMC-based PHA production, and identifies operational aspects that require further optimisation to enhance process robustness and product consistency.

High-silica zeolites are challenging to synthesize in hydroxide media using conventional batch reactors. This study advances beyond traditional batch crystallization methods by exploring a reactor-based strategy for tailoring zeolite properties through mid-way synthesis intervention. By employing the Fed-batch (FB) reactor strategy, we demonstrate that controlled addition of silica precursors at critical synthesis stages, without interrupting temperature or pressure conditions, grants an alternative pathway to high-silica zeolites. Two model systems were investigated: the transformation of FAU-to-CHA and FAU-to-LEV via interzeolite conversion (IZC). These controlled manipulations resulted in an elevated Si/Al ratio and distinct structural properties (e.g., internal silanols) in the final zeolites. The resulting materials were exhaustively characterized and tested in CO2 conversion, highlighting the potential of reactor-based approaches for designing tailored high-performance zeolite catalysts.

This study investigated in situ biomethanation as a biogas upgrading strategy by injecting hydrogen (H2) into anaerobic fed-batch reactors treating wastewater from the pulp and paper industry. Granular sludge was used as inoculum and H2 was supplied at two pressures (0.6 and 0.9 bar overpressure in CB and CC assays, respectively) to evaluate its impact on treatment efficiency, methane (CH4) production, and microbial community dynamics compared to control reactors (CA sets) after an adaptation phase with feeding wastewater only. The CH4 production increased during the first two feeding cycles with H2 supplementation accompanied by a reduction in CO2 emissions. However, this was transient and at the end of cycle 7 acid accumulation (mainly acetic and propionic acids) and reduced CH4 production was observed in both H2-supplemented assays. Microbial community structure changed first as a function of new stirred reactor conditions and later according to amount of H2 addition resulting in three clearly separated groups of communities. The family Syntrophobacteraceae responsible for propionate degradation declined in all reactors due to operational changes and following microbial succession. In control reactors it was out-competed by members of Geobacteraceae and Desulfobulbaceae. In CB and CC assays, Ethanoligenenaceae, Bacillaceae, Kosmotogaceae, Anaerolineaceae, and Anaerolineaceae families were enriched as a result of H2 supplementation. The most abundant methanogens were affiliated to the acetotrophic Methanothrix and the hydrogenotrophic Methanobacterium in all batch reactors. Upon H2 addition the relative abundance of Methanobacterium increased and became predominant in later cycles of CB and CC sets. Despite this shift, both genera coexisted throughout the experiments, suggesting that multiple metabolic pathways contributed to CH4 production under H2-enriched conditions. Although the process demonstrated potential for simultaneous biogas upgrade and wastewater treatment, the overall performance was negatively influenced by increased H2 pressure. This highlights that proper H2 dosing and microbial monitoring are critical to ensure process stability for in situ biomethanation systems. Considering the fragile balance of the investigated wastewater treatment process an ex situ upgrade of biogas using a separate reactor is recommended.

Efficient N-acetylneuraminic acid production by newly discovered cold-adapted N-acetylglucosamine 2-epimerase and N-acetylneuraminic acid lyase via a cold strategy.

To address the limitations of conventional anaerobic digestion (AD), this study explored the integration of microbial electrolysis cells (MECs) with AD to improve biogas production and process stability. While AD is a proven technology for renewable energy recovery from waste, it can suffer from volatile fatty acid accumulation and reduced efficiency. The hybrid MEC–AD system leverages electro-methanogenesis to enhance methane yields and overall system performance. This research evaluated the effects of different electrode materials (graphite plate vs. graphite felt) and applied voltages (0.5 V and 0.7 V) on biogas output, methane content, and operational stability. Results showed that MEC–AD systems significantly outperformed conventional AD, with the highest biogas production reaching 239 ± 3 mL/gVS·d—an increase of up to 162% using graphite felt electrodes at 0.5 V. Internal resistance was also markedly lower with graphite felt (19 Ω/m2) compared to graphite plates (1120 Ω/m2). Furthermore, the pH of the MEC–AD system with graphite felt electrodes was maintained within the optimal range (6.8–7.0), avoiding the acidification seen in control systems. These findings underscore the promise of MEC–AD systems for advancing circular bio-economy initiatives and carbon neutrality. Further work is needed to refine electrode materials and reactor design for improved scalability and efficiency.

Clinical-scale bioreactor production of hiPSC-derived extracellular vesicles modulates miRNA and protein cargo to enhance angiogenic function.

The major green tea polyphenol, epigallocatechin gallate (EGCG), has beneficial antioxidant and anti-inflammatory activities but suffers from poor solubility and stability. Its monoglycosylated derivative, (-)-epigallocatechin gallate 4'-O-α-d-glucopyranoside (EGCG-G1), partially overcomes these limitations. In this study, we engineered Leuconostoc mesenteroides sucrose phosphorylase (LmSPase) for efficient EGCG-G1 production. The triple mutant M3 (T219L/E393I/N335G), created via loop engineering, consensus design, and Rosetta Dock design, exhibited 4.09-fold higher transglycosylase activity at 30 °C, a 1.69-fold longer half-life at 45 °C, and significantly improved regioselectivity compared to the wild type. In a fed-batch reaction at 30 °C and pH 6.0, 25 g/L EGCG was converted within 24 h, producing 31.11 g/L (91.92% yield) of EGCG-G1 with 87.27% purity. This semirational design strategy enhanced the key properties of LmSPase and provides an effective biocatalyst for EGCG-G1 production.

Semi-rational design of a thermostable O-glycosyltransferase from Glycyrrhiza uralensis for efficient conversion of protopanaxadiol.

Raman Spectroscopy (RS) is a powerful technique for the identification of molecules based on the characteristic fingerprint spectra of their vibrational modes. Although challenging, real time spectroscopic monitoring of reactions and processes has great potential value in multiple fields, including process analysis, bio reactors, cell therapies and in vitro metabolomics. Refined chemometrics methodologies are required to datamine the kinetic evolution of multivariate spectral mixtures to establish the constituent reactants and products, as well as the characteristic rates of the reaction. To explore the capabilities and challenges, RS was used to study the chemical kinetics of propyl acetate hydrolysis in an aqueous environment at room temperature, in situ, as a model reaction. The continuous conversion of propyl acetate to 1-propanol and acetic acid was monitored periodically over 250 min using RS with a 532 nm laser source. Simulated admixture solutions, mimicking the reaction from pure reactants to pure products conversion, were also recorded for comparison. Problem based nonlinear least squares (NLS) fitting was applied to both the actual reaction and simulated solution data sets using pure components spectra of both the reactants, propyl acetate, water and products, 1-propanol and acetic acid, in order to visualise and confirm the trends and kinetics of the reaction components. Multivariate Curve Resolution-Alternating Least Squares analysis (MCR-ALS) with kinetic constraints was applied to further resolve the concentration and spectral profiles and to quantify the rates of the reaction. It is demonstrated that MCR-ALS could not accurately resolve the evolving reaction species with respect to concentration, due to rank deficiency. To enhance the analysis, a data augmentation approach was used, seeding the measured datasets with the spectra of the pure components to bias the initial singular value decomposition and spectral unmixing process, resulting in an improved resolution of the systematic variation of concentration dependent data to monitor the kinetic evolution of the reaction mixture. The required seeding weights were optimized by visualizing the sum residual error (SUMR) in least squares fitting of the actual components with the identified pure components by MCR-ALS. Minimum SUMR values for admixtures were found at a seeding weight of 10 000X, while 100X was found to be optimum for the actual reaction. This proof of concept can further pave the way for better analysis and understanding of cascade reactions, and ultimately, potentially of metabolomic pathways.

The effective utilization of crop straw can contribute to sustainable agricultural development. However, how different straw return methods regulate soil fertility and rice yield via bacterial communities in karst paddy fields remains elusive. This field study investigated five straw return treatments [deep plowing (PD); rotary tillage with incorporation (RTM); field rapid composting, (FRC); no-till mulching (NT); and bioreactor (BR)] and a blank control CK (no straw return, fertilizer only) on soil physicochemical properties, bacterial community structure, and rice yield, combined with 16S rRNA sequencing technology. Results indicate the following: (1) all straw incorporation treatments significantly increased soil organic matter (SOM) and nutrient content (p < 0.05), NT and BR treatments increased soil organic matter (SOM) by 38.2 and 36.4%, respectively, compared to CK, while total nitrogen increased by 42.1 and 48.4% with NT; (2) although RTM treatment did not achieve the highest SOM accumulation, it yielded the highest rice yield of 30.37 kg/plot (a significant increase of 13.2% compared to CK), revealing that yield is jointly regulated by soil physicochemical properties and bacterial communities; (3) straw return treatments did not significantly affect bacterial α-diversity (intergroup differences in Shannon index and Chao1 index, p > 0.05), but significantly influenced β-diversity, symbiotic network structure, and community assembly processes. BR treatment formed a complex and stable microbial network structure, while NT exhibited a highly modular community structure (modularity = 0.66); (4) bacterial community assembly under straw return was dominated by deterministic processes, with homogenous selection accounting for 45 and 42% in NT and BR treatments, respectively, significantly higher than CK (28%, p < 0.05); (5) pathwise linear structural equation modeling (PLS-SEM) confirmed that TN (path coefficient 0.97, p < 0.001) and bacterial β-diversity (path coefficient 0.83, p < 0.001) were the most critical factors influencing rice yield. This study elucidates the mechanisms by which different straw return methods drive soil functions by reshaping bacterial community assembly and interaction networks. It provides theoretical support for optimizing straw return technologies in karst paddy fields, such as applying RTM for “yield-priority” scenarios and NT for “Rapid fertilization” scenarios.

Urban areas and suburbs are facing many environmental problems, one of which is the increasing pollution due to the accumulation of cellulosic waste. This article presents a laboratory study on the microbial biodegradation processes of paper as a cellulosic substrate under anaerobic and mesophilic cultivation conditions. A bacterial consortium with cellulose-degrading activity, as well as 4 individual strains originating from a methanogenic anaerobic bioreactor (BRA), was isolated, identified, and characterized. The results demonstrated that the consortium degraded 57.14% of the cellulose matrix within 20 days. Among the individual colonies, colonies 1 and 2 (identified as Clostridium tertium and Agromyces rhizospherae) exhibited lower activities (35.37% and 34.79%, respectively), while colony 3 (Clostridium paraputrificum) displayed the highest activity (83.74%). The mixture of all four colonies achieved lower degradation (21.22%). The performed metagenomic analysis of the microbial consortium revealed a wide variety of different bacterial genera, among which Clostridium, Bacteroides, and Ruminiclostridium dominate, and the species Bacteroides oleiciplenus, Clostridium butyricum, and Ruminiclostridium papyrosolvens. Scanning electron microscopy visualized the adhesion and morphological features of the degrading microbial population. Additional experiments on the development of a laboratory model for the anaerobic biodegradation of cellulose were carried out in BRAs by using different working volumes. A maximal level of cellulose decomposition was achieved in the BRA with a working volume of 1 L, reaching 71.0% cellulose decomposition on day 20. Long-term storage studies confirmed the survival and well-preserved activity of the consortium and individual isolates, demonstrating their potential for the development of bioconversion technologies.

ABSTRACTToday, most recombinant protein drugs are produced by mammalian cells in a stirred‐type bioreactor (BR). Although cell culture scale‐up strategies have been extensively investigated, scale‐up and switching BRs while maintaining comparable culture performance remains a challenging step. This is because the empirical correlations used to determine operating parameters are applicable only for limited situations using similar BRs across scales. In addition, a few small scale‐down models (SSDMs) are able to evaluate cellular sensitivity to the shear environment of manufacturing‐scale BRs. In this study, we focused on the hydrodynamic stress associated with agitation and developed an SSDM that generates high shear stress without undesirable secondary effects such as vortex formation and severe gas hold‐up. In‐house BRs with various scales and configurations were used for fed‐batch culture of CHO‐K1 cells, and their shear environment was characterized by computational fluid dynamics (CFD). Using the dry‐wet approach, we found that average shear stress was well correlated with titer decrease as an indicator of culture performance. We also confirmed that the response to shear stress differs among cell lines, and that evaluation of the shear sensitivity of cells is accordingly a risk mitigation step that is required to ensure successful scale‐up.

Vascular tissue engineering endeavors to design, fabricate, and validate biodegradable and bioabsorbable small-diameter vascular scaffolds engineered with bioactive molecules, capable of meeting the challenges posed by commercial vascular prostheses. A comprehensive investigation of these engineered scaffolds in a bioreactor (BR) is deemed essential as a prerequisite before anyin vivoexperimentation in order to gather information regarding their behavior under physiological conditions and predict the biological activities they may exhibit. This study focuses on an innovative electrospun scaffold made of poly(caprolactone) and poly(glycerol sebacate), integrating quercetin (Q), which is able to modulate inflammation, and gelatin (G), which is necessary to reduce permeability. A custom-made BR was used to assess the performance of the scaffolds maintained under different pressure regimes, covering the human physiological pressure range. As a result, the 3D microfibrous architecture of the scaffolds was notably influenced by the release of bioactive molecules, while retaining the properties required forin vivoregeneration. Furthermore, the scaffolds exhibited mechanical properties comparable to those of native human arteries. The release of Q was effective in counteracting post-surgical inflammation, whereas the amount of released G was adequate to avoid blood leakage and useful to make the material porous during the testing period. This study showcases the successful validation of an engineered scaffold in a BR, supporting its potential as a promising candidate for vascular substitutes inin vivoapplications. Our approach represents a significant leap forward in the field of vascular tissue engineering, offering a multifaceted solution to the complex challenges associated with small-diameter vascular prostheses.

Therapies utilizing human mesenchymal stromal cells (MSCs) are advancing through clinical trials, emphasizing the need for reliable, scalable, and cost-efficient manufacturing processes to support the lot sizes necessary for commercial-scale production. Wharton's jelly MSCs (WJMSCs) are valued for their regenerative abilities and immunomodulatory and anti-inflammatory properties, which contribute to tissue repair. With growing therapeutic demand, the production of WJMSCs must scale to yield billions of cells while maintaining their essential characteristics—identity, purity, and potency—necessary for clinical and regulatory compliance. Achieving such magnitude of expansion entails the utilization of current good manufacturing practice (cGMP)-compliant scalable culture systems that allow bioprocess control and monitoring. This study aimed to establish a scalable serum-/xeno-free expansion process representing a critical step towards a cGMP-compliant large-scale production platform for WJMSC-based clinical applications. Using our in-house GMP-manufactured WJMSCs, which were tested in a Phase Ib clinical trial (NCT03158896), we have previously optimized various culture parameters using a microcarrier (MC)-based three-dimensional (3D) culture system in spinner flasks and demonstrated successful WJMSC expansion. In the present study, we successfully translated culture conditions to a 2 L followed by a STR50 (50 L) stirred-tank bioreactor (BR) (STR), adhering to cGMP requirements. The culture system in the 2 and 50 LBRs supported cell concentrations of approximately 1.2 x 106 cells/mL and attained 24-fold and 27-fold expansion, respectively, with a yield of approximately 37 billion cells in the 50 L culture system after 7 days with a 95% harvest efficiency. Following expansion, WJMSCs preserved their characteristic phenotypes, differentiation potential, chromosomal stability, functional capabilities, and sterility across all tested culture systems. We conclude that the large-scale expansion process of WJMSCs in the STR described herein is highly adaptable to the scale necessary to fulfill the commercial demand for high quality clinical-grade MSCs.

Identification and engineering of a sucrose synthase from Stevia rebaudiana for glycosylation applications.

To achieve the large-scale, low-cost preparation of acetaldehyde lyase (ALS), elastin-like polypeptides (ELPs) as non-chromatographic purification tags were employed to develop an ELP-ALS fusion protein in Escherichia coli. Induction expression results demonstrated that the ELPs tag efficiently improved the soluble expression of the ALS enzyme. Through two rounds of inverse transition cycling (ITC), highly pure ELP-ALS was obtained with an enzyme recovery rate of 85.77%, outperforming Ni2+-affinity chromatography (66.80%). The comparative analysis of enzymatic properties revealed that ELP fusion markedly improved the stability and substrate tolerance of the ALS enzyme. Kinetic parameter analysis under identical conditions showed that ELP-ALS possessed a Vmax of 15.25 U/mg and a kcat/Km of 73.05 s−1·M−1, representing 1.86-fold and 2.97-fold improvements over His-ALS, respectively. Fed-batch reaction using ELP-ALS and acetaldehyde as biocatalyst and substrate, respectively, yielded 95.92 g/L acetoin with 49.32% increase compared to His-ALS (64.24 g/L). These results demonstrated the application potential of ELP-ALS as a promising biocatalyst for acetoin production from acetaldehyde due to its lower preparation cost, higher biocatalytic efficiency, better stability, and substrate tolerance.

Anaerobic co-digestion of poultry litter and poultry slaughterhouse effluent in an anaerobic sequencing fed-batch reactor

ChemDT: A stochastic decision transformer for chemical process control

Aqueous sulfide, a product of the sulfate-reducing process in undissociated form, is a potent inhibitor of methanogens. Furthermore, the biogas generated from the anaerobic digestion of skim latex wastewater (SLW) typically contains > 10,000 ppm H2S. In this study, we investigated the role of zero-valent iron (ZVI) as a sink for both sulfides generated from the reduction of sulfate in SLW and CO2 from acetoclastic methanogenesis. The ZVI-based anaerobic digestion was performed in a fed-batch reactor fed with SLW for 30 days at an organic loading rate (OLR) of 2.34 g COD/L d and a COD/SO42− ratio of around 3. The concentrations of undissociated sulfide in the reactor added with ZVI were 9.1 ± 3.3 mg/L S2−. In contrast, the concentrations of undissociated sulfide in a control reactor were 157.2 ± 44.4 mg/L S2−, which were closer to the levels that can be toxic to methanogens. The ZVI addition promoted the precipitation of iron sulfide and carbonate at a medium pH of 7.9. The biogas generated from the ZVI-based reactor had 94–96% CH4, 4–6% CO2, and undetectable amounts of H2S. However, the methane production in the ZVI-based anaerobic digester decreased by 7.9%, possibly due to the buildup of partial pressure of H2 and subsequent propionate accumulation.

Aerobic treatment of pharmaceutical wastewater was conducted by a microbial consortium (involving four isolated strains: Bacillus pseudomycoides, Bacillus paramycoides, Bacillus timonensis, and Bacillus cereus) to remove a phenolic mixture (4-chlorophenol and catechol). Six parameters were considered regarding the performance of the consortium viz. initial conc. of the mixture, pH of culture media, temperature, volume of culture media, inoculums size, and residence time. Central composite design-based RSM was used at five levels full factorial by utilizing design expert 13.0.5.0. to achieve the highest response i.e., percentage of removal of the mixture. A total of 86 experiments were performed and the outcomes were processed to obtain a second-order polynomial equation along with a design matrix to determine the errors. ANOVA was performed to find out whether the individual independent factors along with the cross-interaction effects of the factors are significant. Justifying the F-value of 13.63, the quadratic model was found to be significant. Optimum conditions were obtained at 1000 mg/L initial conc. of the mixture (500 mg/L each), 8.5 pH value of culture media along with the 600 mL volume of the same, 45 °C temperature, 12% inoculums size (3% inoculums of each of the four bacterial strains) and 48 h of residence time. 99.63% removal of the mixture was obtained by applying these optimized parameters in a batch reactor. HPLC analysis was employed particularly at this point of maximum removal (i.e., center point) to justify the individual removal of 4-chlorophenol and catechol and it was revealed that individually 99.4% 4-chlorophenol and 96.35% catechol were removed from the mixture. To detect the inhibition effect, a kinetic study was deployed and the Haldane Kinetic model validated the present model. Hence, this model can be utilized to remove the mixture from the wastewater as the parameters used here are not pricey so much. Moreover, the incubation time was found to be extensively low while almost complete degradation was gained in case of such a large amount of mixture of two hazardous hydrocarbons together within reasonable temperature and pH.