Resveratrol is a high-value stilbenoid with diverse pharmaceutical and nutraceutical applications. However, its microbial production remains limited by insufficient precursor availability and inefficient carbon utilization. In this study, Escherichia coli was sequentially engineered to establish a methanol-assisted platform for resveratrol biosynthesis. The heterologous pathway was reconstructed by co-expressing Populus tomentosa 4-coumarate: CoA ligase (Pt4CL) and Arachis hypogaea stilbene synthase (AhSTS), followed by introduction of the matBC operon from Streptomyces coelicolor to enhance intracellular malonyl-CoA supply. A synthetic methylotrophic pathway comprising methanol dehydrogenase (mdh2) from Bacillus methanolicus together with 3-hexulose-6-phosphate synthase (hxlA) and 6-phospho-3-hexuloisomerase (hxlB) from Bacillus subtilis was subsequently introduced, and CRISPR interference (CRISPRi) targeting frmA was implemented to modulate formaldehyde metabolism and facilitate methanol utilization. Optimization of heterologous protein expression increased resveratrol production from 7.3 ± 0.2 to 14.1 ± 0.3 mg/L, while matBC-mediated precursor engineering further increased the titer to 20.2 ± 0.4 mg/L. Under glucose-methanol co-feeding conditions, the engineered strain produced approximately 23 ± 0.28 mg/L resveratrol in shake-flask cultivation. Scale-up in a 5-L bioreactor yielded 26.42 ± 0.31 mg/L (132.1 ± 2.4 mg/L total resveratrol) with a PCA conversion efficiency exceeding 95
Oligogalacturonic acid demonstrated potent antibacterial efficacy against foodborne pathogens. To obtain oligogalacturonic acids with favorable antibacterial activity, this study established a controlled enzymatic hydrolysis process for citrus peel pectin based on the characterization of three commercial pectinases. Different fractions of the pectin hydrolysates were then obtained via ethanol fractional precipitation. Results showed that the supernatant from 90
The replacement of cobalt-based siccatives in unsaturated polyester resin curing requires sustainable and scalable biocatalytic alternatives, with fungal peroxidases representing a promising option. For industrial implementation, however, robust large-scale production processes are essential. In this study, we investigated the robustness of a glycerol-based peroxidase production process in K. phaffii driven by the glycerol-derepressible PDF promoter, focusing on industrially relevant bioreactor inhomogeneities. A two-compartment scale-down system was used to mimic substrate and oxygen gradients arising from insufficient mixing and mass transfer at large scale. Substrate inhomogeneity alone led to a moderate reduction in peroxidase titers (approx. 23
Amino acids, as key bio-based chemicals, possess unique physiological functions and chemical properties, making them widely applicable in food additives, animal feed, pharmaceuticals, and cosmetics. However, conventional production methods suffer from inherent limitations, including low conversion efficiency, poor substrate utilization, and significant environmental burdens. The rapid advancement of synthetic biology has provided a revolutionary pathway for amino acid production through the systematic design and engineering of biological systems. This article reviews recent progress in synthetic biology for amino acid biosynthesis, covering the design and reconstruction of metabolic pathways, the development of genetic circuits and dynamic regulation technologies, and the establishment of high-throughput screening platforms. It further elaborates on production strategies for natural amino acids, branched-chain amino acids, and several non-canonical amino acids. On this basis, the key technical applications of synthetic biology in amino acid manufacturing are systematically expounded, and major bottlenecks-such as host cell adaptive evolution and metabolic flux balance-are discussed in the context of current synthetic biology strategies. By integrating and critically reviewing existing studies, this article aims to explore novel research and development strategies to advance the production of biosynthetic amino acids.
Omega-3 polyunsaturated fatty acids (n-3 PUFAs) offer therapeutic benefits, including triglyceride reduction and management of cardiovascular and neurological disorders. This study investigates the use of fish oil processing by‑products, which consist mainly of fatty acid methyl and ethyl esters, as novel substrates for n‑3 PUFAs production. To overcome the low efficiency of conventional chemical methods and the vulnerability of free lipase to alcohol toxicity and poor reusability, we developed a stable immobilized lipase system. The target enzyme (CALB) was immobilized onto macroporous resin LXTE‑1000 via adsorption‑crosslinking, and optimal hydrolysis conditions were determined. The resulting immobilized enzyme (LXTE‑1000@CALB) showed markedly enhanced thermal and operational stability, retaining approximately 64.21
Cyanophycin granule polypeptide (CGP), also known as multi-l-arginyl-poly(aspartic acid), is a biodegradable biopolymer composed primarily of aspartic acid and arginine. Due to its versatile functional properties, CGP has attracted increasing interest for potential applications in food, medicine, cosmetics, agriculture, and corrosion inhibition. The objective of this study was to biosynthesize a biologically derived corrosion-inhibiting biomaterial using recombinant Escherichia coli BL21(DE3) expressing cyanophycin synthetase (CphA), followed by cost-effective induction strategy. Specifically, this work aimed to establish a high-cell-density cultivation process capable of achieving improved CGP production while reducing dependence on costly inducers such as IPTG. Initial shake-flask experiments demonstrated that lactose induction resulted in higher CGP production and biomass formation compared to IPTG induction. In addition, supplementation with phosphate, ribose, trace elements, yeast extract, and tryptone further improved CGP accumulation. Based on these findings, a high-cell-density fed-batch fermentation strategy using lactose as both inducer and carbon source was developed, maximum gravimetrically recovered crude soluble and insoluble CGP-containing fractions of 35.4 and 17.8 g/L. Product characterization was further supported by FTIR, XRD, MALDI-MS. Furthermore, the recovered CGP-related material was evaluated in preliminary corrosion inhibition experiments under acidic conditions and showed significant reduction in corrosion rates compared with untreated control samples, indicating its potential for future corrosion-protection applications.
Facing the dual challenges of swine manure pollution control and carbon resource recovery under intensive farming conditions, conventional anaerobic fermentation technologies are limited in their application for high-value conversion due to low hydrolysis efficiency and high operational costs. To address these limitations, this study proposed a self-heating strategy using short-term aerobic pretreatment, aiming to optimize substrate pretreatment and enhance the synthesis of short-chain fatty acids during subsequent acidogenic fermentation. The results demonstrated that this pretreatment approach rapidly activates aerobic microbial metabolism and significantly improves the substrate's overall properties, as reflected by a decreased pH and increased conductivity, moisture content, total protein, and total organic carbon, and effectively promotes the solubilization and hydrolysis of organic matter, thereby increasing the bioavailability of substrates for anaerobic fermentation. Microbial community analysis further revealed that the pretreatment process selectively enriched hydrolytic-acidogenic bacteria, including Clostridium, Bacillus, Paenibacillus, and Streptomyces, while synergistically enhancing the activities of key metabolic enzymes such as cellulase, protease, and acetate kinase. These effects systematically strengthened the metabolic pathways from substrate degradation to acid synthesis. As a result, the short‑term aerobic pretreatment achieved a significant improvement in SCFAs production and energy efficiency. Specifically, the total SCFAs yield increased by 21.31
In the present work, the marine bacterium Vibrio alginolyticus Mo245 demonstrated the ability to secrete an extracellular polysaccharide (EPS) concomitant with the intracellular accumulation of a polyhydroxyalkanoate (PHA). Upon cultivation in glycerol as the sole carbon source, V. alginolyticus Mo245 co-produced a glycosaminoglycan (GAG), named EPS-Mo245, and the homopolymer poly(3-hydroxybutyrate) (P3HB), attaining maximum concentrations of 1.29±0.01 g/L and 1.78±0.00 g/L, respectively, within a 24 h cultivation. EPS-Mo245 was a hyaluronic acid (HA)-like GAG, composed of glucuronic acid (40.5±2.2 mol
Rapid generation of monoclonal CHO cell lines remains a key bottleneck in mammalian cell line development (CLD), often requiring months of iterative screening and cloning. Here, we present an integrated droplet microfluidic workflow for high-throughput screening and isolation of antibody-producing ExpiCHO-S cells using a FRET-based secretion assay. The FRET assay was developed and optimized for picodroplet-based screening by evaluating probe configuration, donor-to-acceptor ratio, antibody concentration range, and signal saturation behavior. The platform enables picodroplet-based detection of secreted antibodies, sequential enrichment of antibody-producing cells, and image-verified single-cell isolation. Application of this approach yielded image-verified monoclonal antibody-producing clones that maintained consistent antibody production during the stability assessment performed in this study, including in the absence of selection pressure. By reducing the early clone-identification phase from DNA transfection to validated 96-well antibody-producing clones to approximately 5 weeks, this workflow addresses key limitations of conventional approaches and provides both a practical FRET assay-development framework and a scalable strategy for streamlining early CLD workflows in biomanufacturing.
Production of Fab antibody fragments with Escherichia coli strains engineered for extracellular product release can reduce downstream processing requirements but often compromises cellular integrity, resulting in increased cell lysis. To investigate the balance between product formation, product release, and cellular robustness, 32 fed-batch cultivation conditions spanning six process parameters were systematically evaluated in automated high-throughput bioreactors. In total, 100 cultivations were performed, and mechanistic modeling was applied to estimate cell lysis, product formation, and product release kinetics. The combined experimental and model-based analysis revealed a pronounced trade-off between productivity and robustness. Conditions yielding high Fab titers showed minimal cell lysis rates but also slow product release, whereas elevated release kinetics were consistently associated with substantial lysis and reduced Fab titers. Induction strength emerged as the primary driver for maximizing Fab titer, while production temperature governed the balance between product release and cell lysis. Compared to the reference condition, Fab titers increased approximately twofold to 730 mg L ^-1 while reducing the cell lysis rate by up to eightfold to near-negligible levels, albeit at the expense of a 2.2-fold lower product release rate. Robustness studies under large-scale–relevant conditions identified moderate substrate heterogeneities as an additional factor enhancing productivity while reducing cell lysis. Furthermore, the extensive dataset enabled refinement of a previously developed product model by introducing an induction-strength-dependent production load and provided mechanistic insight into the relationship between product release and cell lysis. Overall, the study demonstrates how mechanistic modeling enhances the interpretation of automated high-throughput screening experiments by linking observable process responses to underlying physiological behavior and enabling quantitative analysis of complex production trade-offs.
The rapid pace of urbanization and increase in population have resulted in an unprecedented increase in the generation of municipal solid waste (MSW), highlighting the urgent need for sustainable and effective waste management approaches. Composting stands out as an environmentally friendly approach for the valorization of organic waste among the diverse techniques available. This paper provides a critical analysis of the essential phases involved in MSW composting, starting with preprocessing methods and highlighting the significance of source separation in improving the quality of compost. This paper presents a comparative analysis of major composting methods, specifically windrow, aerated static pile, and in-vessel systems, focusing on their efficiency and operational control. Furthermore, the significant process variables, including temperature, pH, C/N ratio, particle size, moisture content, aeration rate, and the shape of windrows, were discussed. This review encompasses a range of maturity and stability indices utilized to determine compost quality and its readiness for its application. The integration of process control is highlighted as a crucial factor for optimizing composting outcomes. This paper also synthesizes recent advances and challenges in MSW composting, providing a thorough understanding that offers insights for enhancing current practices and directing future research efforts. Important results from research on various kinds of composting processes, particularly aerobic composting, since it is a feasible technique for the composting of MSW; monitoring parameters; the Indian context; and compost utilization are described in this paper.
Chemico-biological cascade catalysis of hexose into 2,5-bis(hydroxymethyl)furan (BHMF) is desirable but limited by the incompatibility between the solvent and whole-cell catalyst. In this work, aqueous polyethylene glycol (PEG-1000) solution was proven to facilitate both chemical dehydration of fructose and bioreduction of 5-hydroxymethylfurfural (HMF). A high HMF yield of 0.80 was obtained from fructose using 50 mol
The growing industrial demand for economically attractive large-scale bioprocesses has increased the need for reliable and computationally feasible modelling approaches. Gas fermentation plays a particular role as the targeted commodities ask for large-scale solutions using bubble-column type bioreactors that allow them to benefit from the economy-of-scale. To reduce development efforts, large-scale designs increasingly build on computational fluid dynamics (CFD) simulations. State-of-the-art approaches employ finite-volume Reynolds-averaged Navier-Stokes (FV-RANS) methods that require enormous computational efforts. In contrast, Euler-Lagrange (E-L) approaches offer detailed local resolution as a prerequisite for optimum bioreactor design. By tracking individual bubbles, they predict bubble population dynamics while computations are parallelized. However, to cope with realistic bubble numbers, large-scale simulations typically make use of the so-called ‘parcel size’ approach, i.e. multiple bubbles are represented by a single computational particle. This study applies Lattice Boltzmann large eddy simulations (LB-LES) to illustrate the observed consequences: Swarm-like bubble behavior is found that results in inhomogeneous bubble distributions. As a consequence, the volumetric mass transfer coefficient kLa, a key criterion for design, may be biased. Comparing results with state-of-the-art FV-RANS simulations revealed similarities of transient flow structures and averaged velocity values. However, local deviations are found that mirror handling differences of turbulence representation and the impact of the parcel approach. The findings demonstrate the potential and current limitations of parcel-based modelling for large-scale bioreactors.
This study pioneers the investigation of the fundamental mechanisms by which bamboo biochar (BBC) enhances volatile fatty acids (VFAs) production from cornstalk through anaerobic fermentation by rumen microorganism (RM). The results demonstrate that BBC prepared at 450 °C significantly boosts VFAs production in anaerobic fermentation of cornstalk by 43.8
Antibiotic stress severely inhibits anaerobic digestion performance by disrupting microbial metabolism and volatile fatty acid (VFA) conversion. To address this bottleneck, a sulfate (SO42−)-assisted anaerobic digestion system co-coupled with waste sludge-derived biochar and zero-valent iron (BC/Fe0) was developed to treat livestock wastewater under antibiotic stress. Results demonstrated that the coupled system exhibited exceptional process robustness. Even under a severe shock load of 100 mg/(L·d) antibiotic, the system maintained a chemical oxygen demand (CODCr) removal efficiency of 76.5 ± 1.6
In bioprocess engineering, model-based methods play a vital role in understanding complex dynamics of novel species or strains. However, the model development is often hampered by prohibitive costs associated with redundant reference analyses and poorly informed sampling schedules due to insufficient prior knowledge about the process dynamics. We propose a model-free PAT strategy, termed Raman-guided sample subset selection (RGSS) to prioritise informative offline reference assays from inline Raman spectra before nonlinear model calibration. RGSS is demonstrated with Saccharomyces cerevisiae fed-batch fermentations and evaluated by auto- and cross-validation (CV), as well as with practical identifiability analysis of an unstructured mechanistic model. The approach is implemented using constrained vector quantization (CVQ) with full-spectrum and analyte-specific wavenumber-selection and benchmarked against uniform-in-time sampling, random subsampling, and Kennard–Stone (KS) selection. The best RGSS scenarios, using only five selected reference assays per analyte, retained offline CV accuracy (NRMSE = 3.49) close to the full-data reference (NRMSE = 3.29) using all 22 samples. KS selection was also competitive for ten selected samples (NRMSE = 3.79), whereas uniform-in-time sampling resulted in higher CV errors for five and ten samples (8.47 and 65.57). Random subsampling occasionally produced competitive subsets, but showed broad variability over 20 random runs (NRMSE median [IQR]: 6.14 [4.84–47.76] and 15.48 [5.07–44.34]) for five and ten randomly selected samples. These results support RGSS as a cost-efficient, spectrally informed sample subset selection framework for prioritising offline assays in model-based bioprocess development.
Biomethane is a clean, renewable, and eco-friendly unconventional natural gas resource that has attracted widespread global attention. However, the differences in biomethane generation and the constraining factors under the drive of indigenous versus exogenous microorganisms remain unclear. To address this, anaerobic fermentation experiments simulating coal‑derived biomethane generation were conducted using two distinct microbial sources: indigenous microorganisms enriched from fresh coal samples from the study area and exogenous microorganisms optimized under laboratory conditions. Five representative coal samples from the Wuguantun and Baode mining areas were used as carbon substrates. The efficiency of biomethane production was evaluated based on gas chromatography and analysis using four kinetic models. By integrating methods including coal petrographic and proximate analyses, 16 S rRNA high-throughput sequencing, and Fourier transform infrared spectroscopy, principal component analysis and metabolic pathway analysis were applied to systematically elucidate the main controlling factors and synergistic mechanisms governing biomethane generation. The results indicate that although both indigenous and exogenous microorganisms follow a similar three‑stage process during coal‑degrading methanogenesis, their gas production efficiencies differ significantly. Bioaugmentation with exogenous microbial consortia systematically optimized the biomethane generation process, increasing the maximum methane potential (A0) by approximately 80
Lignocellulosic biomass represents a sustainable energy source as ethanol fuel demand increases; process integration and efficient C6/C5 sugars utilization are essential for achieving techno-economically viable production. Here, alkaline-sulfite pretreatment combined with disc refining was applied to sugarcane bagasse for conserving most of cellulose and hemicellulose fractions. Then, β-glucosidase supplementation of cellulase at rational enzyme dosage (18 FPU/g biomass) was established for the enzymatic hydrolysis of the pretreated solids, achieving approximately 75 Q_P = 3.25 g L− 1 h− 1) than Saccharomyces cerevisiae PE-2 ( Q_P = 2.17 g L− 1 h− 1). Notably, Scheffersomyces stipitis Y-7124 was the only strain capable of significantly consuming xylose, resulting in 46
Microalgal CO₂ fixation is a promising strategy for industrial carbon capture, yet its application remains limited by inefficient light utilization. Although the flashing light effect has been explored as a light-management strategy for improving photon utilization in microalgal cultivation, its effectiveness depends on spectral composition, flashing frequency, duty cycle, and cultivation conditions. In this study, light quality, including red (R), blue (B), white (W), and mixed red-blue (RB) light, and PWM flashing parameters, including frequency (10–1000 Hz) and duty cycle (30
