Bioprocess and Biosystems Engineering最新文献

Experimental models for exploring abnormal brain blood vessels, including ischemic stroke, are crucial in neuroscience; recently, significant attention has been paid to artificial tissues through tissue engineering. Nanofibers, although commonly used as tissue engineering scaffolds, undergo structural deformations easily, making it challenging to create uniform tissue, especially for the smallest-diameter ones such as perforating arteries. This study focused on the development of a platform capable of reconstructing structurally and functionally replicated perforating arteries. To ensure structural consistency, 3D-printed modules were developed to minimize the structural deformation of nanofibrous scaffolds when integrated into a 3D-printed vessel culture dish. Surface structures and physical characteristics of the nanofibers before and after installation were compared using scanning electron microscopy, contact angle analysis, surface area analysis, and universal testing machine (UTM) analysis. The results showed a uniform thickness distribution, topography, maximum load, tensile strain, tensile strength, surface area, pore size, and pore volume of the nanofibers. For consistency in tissue culture, smooth muscle, endothelial, and astrocyte cells were co-cultured by continuously measuring the pH of the medium and replenishing the depleted glucose using the Kalman filter control system. The functional efficacy and consistency of the artificial perforating vessels were confirmed under oxidative stress induced by exposure to hydrogen peroxide. Transcriptional mRNA expression trends were similar to those in vivo for antioxidant enzymes, neurotrophic factors, inflammatory factors, and endothelial cell activation factors, with very low variation between tissues. This study provides a research platform for studying the oxidative stress environments related to stroke by mass-producing perforating arteries with consistent structures and functions.

Lipases are one of the ubiquitous enzymes that belong to the hydrolases family and have a wide variety of applications. Cold-active lipases are of major attraction as they can act in lower temperatures and low water conditions because of their inherent greater flexibility. One of the novel applications of lipase is the enrichment of ω-3 polyunsaturated fatty acids (PUFA) in plant and fish oils. This study is aimed at the isolation and identification of cold-active lipase producing bacterium from marine sources, preliminary optimization of medium constituents and conditions, purification of lipase using chromatographic techniques, biochemical characterization, and ultimately the exploration of its application in the enrichment of ω-3 PUFA in flax seed oil. Psychrobacter alimentarius ILMKVIT was identified as the potential cold-active lipase producing bacterium based on its lipolytic activity in rhodamine B agar, titrimetric, and p-nitrophenyl palmitate (p-NPP) assays. One factor at a time (OFAT) analysis, revealed, an incubation time of 4.5 days, alkaline pH of 9, the temperature of 25 °C, peptone, and yeast extract as nitrogen sources, olive oil as inducer sources, 1% inoculum size, and NaCl as mineral sources as optimum production medium constituents and conditions for lipase production. Lipase purification was achieved by ion exchange and gel-filtration chromatography with a 9.27% yield and 37.51-fold purification. Biochemical characterization reported that the lipase is cold-active, alkaline, enhanced by Fe³⁺ metal ions, and tolerant to organic solvents, detergents, and inhibitors. P. alimentarius ILMKVIT lipase-hydrolysis followed by urea complexation of flax seed oil resulted in the enrichment of ω-3 PUFA, especially α-linolenic acid (ALA). Hence, the novel cold-active lipase from P. alimentarius ILMKVIT could be used to enrich ω-3 PUFA in flax seed oil and developed further as a prominent nutrient supplement for health benefits.

Microbial fermentation is an effective method to improve the functional activity of oats (Avena sativa L.), while there are some limitations to the advantages of single microbial fermentation. In this study, a microbial co-culture fermentation system with Monascus anka, Saccharomyces cerevisiae and Bacillus subtilis to release and conversion oat phenolics was established. Results showed that the optimal microbial co-fermentation system was obtained by adding Saccharomyces cerevisiae on the fourth day and Bacillus subtilis on the eighth day during Monascus anka fermentation (MF + 4S + 8B). The phenolic content was reached 26.93 mg GAE/g DW, which increased 41.08 times compared to un-fermented oats (UF). In the process of co-fermentation systems, cellulase and β-glucosidase (r² = 0.97, p < 0.01) had a positive correlation with the release of phenolics. SEM combined with HPLC showed that the complex enzyme system produced by microbial co-fermentation enhanced the disruption of oat cell structure, as well as altered the phenolics fractions and facilitated the conversion of bound phenolics to free phenolics, especially the content of chlorogenic acid and vanillic acid in the free forms was increased 31.42 and 14.15 times, respectively. Additionally, the phenolic contents were increased and the components were changed with the microbial co-fermentation of crude enzyme solution further added, which validated the positive influence of complex enzyme system of MF + 4S + 8B in the phenolic release and transformation of oats. Therefore, this study systematically investigated the phenolic mobilization in oats during the co-fermentation period, which provides a viable option for improving the functional properties of cereal products, as well as the application of microbial cell factories.

Levilactobacillus brevis KCL010 was fermented in a simple medium containing 8% (w/v) of rice bran extract. We modified the carbon, nitrogen, and initial pH conditions using 10 g/L of sucrose, 10 g/L of yeast extract, and 5.0 of pH, respectively. To minimize the pH increase due to decarboxylation, we fermented 100 mL of modified synthetic medium containing citrate-phosphate buffer (CPB, pH 5.0) of 25-200 mM in 250 mL Erlenmeyer flasks. After 72 h of fermentation with 50 mM CPB, the maximum GABA concentration and conversion efficiency were 3.42 g/L and 22.39%. Furthermore, the potential α-glucosidase inhibitory activity, MTT assay, and oil red O staining were determined by fermented extracts of L. brevis KCL010. At the highest concentration of 500 μg/mL, the α-glucosidase inhibition percentages for non-fermented rice bran (NFRB), rice bran fermented by L. brevis (RBFL), and GABA (analytical standard) extracts were 55.03%, 58.37%, and 59.48%, respectively. All extracts exceeded 80% viability, suggesting that there was no cytotoxic to 3T3-L1 adipocytes. The rice bran fermented by L. brevis (RBFL) extract shows a high inhibition of lipid accumulation by 29.33% compared to those of extracts.

Cyanobacterial exopolysaccharides (EPS) remain released by cyanobacteria in the surrounding environment with the main purpose of protection against harmful environmental conditions. Recently, they have received significant attention due to their unique structural characteristics, functional properties, and potential applications across various fields. The current study describes the evaluation of EPS production under salinity stress from Arthrospira maxima. The application of high salinity up to 40 g/L enhanced EPS production, which was collected and purified by alcohol precipitation followed by membrane dialysis and lyophilization. A yield of 60 mg/L was obtained. The Size exclusion chromatography gave for the purified EPS an apparent molecular weight of 2.1 × 10⁵ Da. Monosaccharide composition showed that EPS is a heteropolymer, with mannose, xylose, and glucuronic acid identified as the predominant monosaccharides and derivatives. Nuclear magnetic resonance spectroscopy (¹³C and ¹H) confirmed that EPS is a heteropolysaccharide, entirely in α- anomeric configuration, with glucuronic acid as a main monomer that is probably linked to mannose and xylose via α-glycosidic linkages. Bioactivity assessment of EPS revealed that it exhibits antibacterial activity against several strains, notably, Bacillus subtilis (MIC: 0.6 ± 0.05 mg/mL), Bacillus cereus (MIC: 1 ± 0.01 mg/mL), Escherichia coli (MIC: 0.8 ± 0.01 mg/mL) and Klebsiella pneumonia (MIC: 0.8 ± 0.01 mg/mL). Antioxidant activity was measured using the DPPH radical scavenging assay, yielding an IC₅₀ of 6.83 mg/mL. Besides, EPS was also found to exhibit an interesting emulsifying property with several oil types, indicating its potential as a versatile biopolymer for applications in various industrial sectors.

The transition towards sustainable bioprocesses requires renewable feedstocks to reduce dependency on finite resources. While plant-based feedstocks offer significant potential, their complex composition poses new challenges. The microorganisms often exhibit polyauxic growth when presented with multiple carbon sources simultaneously, consuming them in a distinct order according to their carbon source preferences. The traditional investigation of polyauxic growth involves laborious sampling and offline analysis, hindering high-throughput screenings. This study introduces an efficient method for identifying carbon source consumption and their order of metabolization by various microorganisms using the respiration activity monitoring system (RAMOS) in shake flasks. As aerobic carbon metabolization and oxygen consumption are strictly correlated, the characteristic phases of polyauxic growth are visible in the oxygen transfer rate (OTR) and can be assigned to the respective carbon sources. An extended 16-flask RAMOS enables real-time monitoring of microbial respiration on up to seven carbon sources and one reference cultivation simultaneously, thus providing crucial insights into their metabolization without extensive sampling and offline analysis. The method's accuracy was validated against traditional high-performance liquid chromatography (HPLC). Its applicability to both fast-growing Escherichia coli (investigated carbon sources: glucose, arabinose, sorbitol, xylose, and glycerol) and slow-growing Ustilago trichophora (glucose, glycerol, xylose, sorbitol, rhamnose, galacturonic acid, and lactic acid) was demonstrated. Additionally, it was successfully applied to the plant-based second-generation feedstock corn leaf hydrolysate, revealing the bioavailability of the included carbon sources (glucose, sucrose, arabinose, xylose, and galactose) and their order of metabolization by Ustilago maydis.

Animal manure is considered to have great potential for phosphorus (P) recovery due to its high P content, while P recovery is limited by the transfer of P from the solid phase to the liquid phase. The conventional dissolution process by adding chemical acid reagents is not economically feasible for animal manure. This study used food waste (FW) as a co-substrate for the anaerobic fermentation of pig manure (PM) to achieve the release of P. The operational parameters were optimized, and the mechanisms of acidification and P release were further studied. The results showed FW promoted lactic acid production and rapid acidification. As FW increased from 0 to 80%, the concentrations of lactic acid rose from 0.12 ± 0.04 to 11.95 ± 1.37 g/L, with pH decreasing from 7.55 to 4.43. The ratio with FW/PM = 1:2 was the optimal condition, which led to the highest soluble phosphate concentration (350.39 ± 8.59 mg/L) in 72 h, with a TP release rate of 74.24 ± 1.81%. Multiple regression analyses established key relationships to predict pH changes in the reactor.

A first principle soft-sensor for biomass and substrate estimation in upstream bioprocessing based on the fusion of elemental balancing and nonlinear kinetics is presented. It aims to extend the validity range of well-established elemental balancing soft sensors to substrate saturated and overfeeding conditions that often occur in induced production phases. An experimental study with recombinant E. coli cultivations was conducted to illustrate the soft-sensor principle and to analyze the accuracy as well as generalizability of the approach. Under substrate limited growth the extended soft-sensor showed similar performance as classical elemental balancing. In induced production phases however, a decline in maximum substrate uptake capacity ( $q_{\mathrm{Smax}}$ ) of up to 80% was observed, where the extended soft-sensor showed up to 41 % better estimates for the biomass and up to 75 % better estimates for the substrate in terms of NRMSE. The paper discusses the possible benefits as well as the requirements for the implementation of the extended elemental balancing soft-sensor.

This research provides an important approach for low-nitrogen wastewater treatment through anaerobic ammonium oxidation (Anammox), and Anammox granule sludge (AnGS) in the Upflow. Blanket Filter Anammox (UBFA) system through shortening the hydraulic retention time was successfully cultivated. The percentage of medium granules (1.0-2.0 mm) with the highest Anammox activity increased from 0 to 28.5%, and the proportion of flocs (0-200 μm) reduced from 84.5% to 17.6%. Through the multidimensional analysis of AnGS, the relationship between AnGS and EPS secretion, low SVI, high PN/PS, multiple filamentous bacteria, and AnAOB were explored. Microelectrode tracing tests demonstrated that the main anammox reaction active layer was 0-1500 μm, and the highest activity was observed at 200-400 μm, whereas denitrification activity and N₂O production were mainly distributed in the granules deep layer of 1500-2500 μm. The research showed that Candidatus Brocadia and Candidatus Kuenenia were the predominant anammox species in the UBFA system, while the abundance of AnAOB was higher in medium granules.

Heavy crude oil reserves are characterized by their high viscosity and density, largely due to significant quantities of asphaltenes. The removal of asphaltene precipitates from oil industry installations is crucial, as they can contaminate catalysts and obstruct pipelines. Therefore, this study aimed to bio-transform heavy oil asphaltenes into smaller molecules using the yeast Yarrowia lipolytica, known for its ability to efficiently degrade hydrophobic substrates. For this purpose, asphaltenes were extracted from crude oil samples, and yeast growth was assessed in a mineral medium containing 2, 5, or 10 g L^-1 of asphaltenes. After 168 h of incubation, liquid-liquid extraction was conducted on samples from the Yarrowia lipolytica growth medium using chloroform. The extracted fractions were then quantified by gas chromatography. The results indicated that the yeast could utilize the asphaltenes as a carbon source for growth, though there was a delay in growth compared to the control (glucose as the carbon source), with a maximum biomass concentration of 2.26 g L^-1 achieved at 144 h. From the experimental design, it was determined that a higher concentration of aromatic compounds was achieved under the conditions of 115 rpm, 2 g L^-1 of asphaltenes, and 0.5 g L^-1 of cell inoculum. Conversely, to obtain a higher concentration of saturated compounds, the optimal conditions were 160 rpm, 5 g L^-1 of asphaltenes, and 1.0 g L^-1 of cell inoculum. Molecular docking results indicated that asphaltenes have a high affinity for cytochrome P450, laccase, and Lip2, with interactions observed with their catalytic triads, suggesting a significant role for these enzymes in asphaltene bioconversion.