Establishment of a semi-continuous nano-production line using the Microfluidizer® technology for the fabrication of lipid-based nanoparticles part 1: Screening of critical parameters and design of experiment optimization studies.

A variety of strategies for producing high-quality nanoparticles have been reported in recent years. Batch-based bottom-up and top-down technologies are generally the most efficient methods, but present a number of challenges, particularly in terms of variability, safety, sustainability and large-scale production. In this study, a scalable, semi-continuous production line was built by connecting individual processing units, including a high shear mixing device, the Microfluidizer® technology and a cooling system. Each unit was equipped with an adequate temperature control to allow solvent-free production of solid lipid nanoparticles (consisting of Precirol® ATO 5 or Gelucire® 43/01) and nanostructured lipid carriers (additionally comprising Labrafac™ lipophile WL 1349). Subsequently, critical formulation parameters and critical process parameters (CPPs) of the individual processing units and their effects on particle size (i.e., critical quality attribute (CQA)) were investigated to identify appropriate input parameters for the subsequent Design of Experiment (DoE) studies conducted after linking the process units to a semi-continuous production line. For particle size monitoring, spatially resolved dynamic light scattering (SR-DLS) measurements were conducted and compared to standard DLS measurements to evaluate the applicability of SR-DLS as an inline monitoring tool. It was found that matrix composition, emulsifier concentration, pressure and number of cycles when processing through Microfluidizer® processor were the most influencing parameters. By optimizing these parameters, five-times higher throughputs could be achieved by the semi-continuous manufacturing line. In addition, the particle size measurements with SR-DLS confirmed the feasibility of implementing this technology for real-time particle size monitoring as an important safety factor in quality control.

High efficiency production of 5-hydroxyectoine using metabolically engineered Escherichia coli.

The 5-hydroxyectoine is a natural protective agent with long-lasting moisturising and radiation resistance properties. It can be naturally synthesized by some extremophiles using the "bacterial milking" process, but this can corrode bioreactors and downstream purification may cause environmental pollution. In this study, an engineered Escherichia coli (E. coli) strain was constructed for the 5-hydroxyectoine production. First, three ectoine hydroxylases were characterised and the enzyme from Halomonas elongata was the most effective. The L-2,4-diaminobutyrate transaminase mutant was introduced into the engineered strain, which could accumulate 2.8 g/L 5-hydroxyectoine in shake flasks. By activating the glyoxylate cycle and balancing the α-ketoglutarate distribution, the 5-hydroxyectoine titer was further increased to 3.4 g/L. Finally, the optimized strain synthesized 58 g/L 5-hydroxyectoine via a semi-continuous feeding process in a NaCl-free medium. Overall, this study reported the highest titer of 5-hydroxyectoine synthesized by E. coli and established a low-salt fermentation process through the aforementioned efforts.

Self-flocculating Spirulina platensis CMB-02 to efficiently treat ammonia nitrogen of rare earth elements wastewater.

The study aimed to evaluate the cyanobacteria Spirulina platensis CMB-02 (S. platensis CMB-02) with self-flocculation properties to treat the ammonia nitrogen of rare earth elements (REEs) wastewater. The results demonstrated that S. platensis CMB-02 could effectively remove total ammonia nitrogen (TAN) and total inorganic nitrogen within 5 days. Simultaneously, a self-flocculation efficiency of 82.59 % was achieved by microalga in 30 min after wastewater treatment. The pH, tightly bound extracellular polymeric substances (TB-EPS), and cell morphology of S. platensis CMB-02 were identified as key factors influencing its self-flocculation capabilities. Moreover, the established semi-continuous process with a 20 % renewal rate showed a stable treatment effect, representing a TAN degradation rate of 10.9 mg/(L·d). These obtained findings could conclude that the developed approach mediated with self-flocculating S. platensis CMB-02 was a promising way for REEs wastewater treatment.

Establishment of a semi-continuous scale-down clone screening model for intensified perfusion culture.

PURPOSE: Perfusion cultures have been extensively used in the biotechnology industry to achieve high yields of recombinant products, especially those with stability issue. The WuXiUP™ platform represents a novel intensified perfusion that can achieve ultra-high productivity. This study describes a representative scale-down 24-deep well plate (24-DWP) cell culture model for intensified perfusion clone screening. METHODS: Clonal cell lines were expanded and evaluated in 24-DWP semi-continuous culture. Cell were sampled and counted daily with the aid of an automated liquid handler and high-throughput cell counter. To mimic perfusion culture, 24-DWP plates were spun down and resuspended with fresh medium daily. Top clones were ranked based on growth profiles and productivities. The best performing clones were evaluated on bioreactors. RESULTS: The selected clones achieved volumetric productivity (Pv) up to 5 g/L/day when expressing a monoclonal antibody, with the accumulative harvest Pv exceeding 60 g/L in a 21-day cell culture. Product quality attributes of clones cultured in 24-DWP were comparable with those from bioreactors. A high seeding strategy further shortened the clone screening timeline. CONCLUSION: In this study, a 24-DWP semi-continuous scale-down model was successfully developed to screen for cell lines suitable for intensified perfusion culture.

A Novel Bio-Purification Process Employing an Engineered E. coli Strain for Downstream Processing of Lactic Acid Solutions from the Fermentation of Agro-Industrial by-Products.

This study aims to integrate a novel bio-purification process employing an engineered E. coli strain in the downstream processing of lactic acid (LA) fermentation broths from low-cost renewable biological feedstocks. Fermentation broth of candy waste and digestate mixture was used as a real biological feedstock. An engineered E. coli strain that selectively catabolize impurities without catabolizing LA was initially adapted on the biological feedstock, followed by shake flask experiments to prove the bio-purification concept. Scale-up and validation in a bench-scale bioreactor followed, before developing a semi-continuous membrane bioreactor (MBR) bio-purification process. The MBR bio-purification was assessed with biological feedstocks which simulated ultrafiltration or nanofiltration permeates. Incomplete removal of impurities and increased fouling was observed in the case of the ultrafiltration permeate. Contrarily, the nanofiltration permeate was successfully treated with MBR bio-purification, since low membrane fouling, 100% maltose and acetic acid removal, and no LA catabolism was achieved. MBR bio-purification as a post-treatment step in the downstream processing of LA was demonstrated as a promising technology for increasing the purity of LA solutions.

Semi-continuous cultivation of EPS-producing marine cyanobacteria: A green biotechnology to remove dissolved metals obtaining metal-organic materials.

Given the necessity for bioprocesses scaling-up, the present study aims to explore the potential of three marine cyanobacteria and a consortium, cultivated in semi-continuous mode, as a green approach for i) continuous exopolysaccharide-rich biomass production and ii) removal of positively charged metals (Cu, Ni, Zn) from mono and multi-metallic solutions. To ensure the effectiveness of both cellular and released exopolysaccharides, weekly harvested whole cultures were confined in dialysis tubings. The results revealed that all the tested cyanobacteria have a stronger affinity towards Cu in mono and three-metal systems. Despite the amount of metals removed per gram of biomass decreased with higher biosorbent dosage, the more soluble carbohydrates were produced, the greater was the metal uptake, underscoring the pivotal role of released exopolysaccharides in metal biosorption. According to this, Dactylococcopsis salina 16Som2 showed the highest carbohydrate productivity (142 mg L-1 d-1) and metal uptake (84 mg Cu g-1 biomass) representing a promising candidate for further studies. The semi-continuous cultivation of marine cyanobacteria here reported assures a schedulable production of exopolysaccharide-rich biosorbents with high metal removal and recovery potential, even from multi-metallic solutions, as a step forward in the industrial application of cyanobacteria.

The Feasibility of Semi-Continuous and Multi-Frequency Thoracic Bioimpedance Measurements by a Wearable Device during Fluid Changes in Hemodialysis Patients.

Repeated single-point measurements of thoracic bioimpedance at a single (low) frequency are strongly related to fluid changes during hemodialysis. Extension to semi-continuous measurements may provide longitudinal details in the time pattern of the bioimpedance signal, and multi-frequency measurements may add in-depth information on the distribution between intra- and extracellular fluid. This study aimed to investigate the feasibility of semi-continuous multi-frequency thoracic bioimpedance measurements by a wearable device in hemodialysis patients. Therefore, thoracic bioimpedance was recorded semi-continuously (i.e., every ten minutes) at nine frequencies (8-160 kHz) in 68 patients during two consecutive hemodialysis sessions, complemented by a single-point measurement at home in-between both sessions. On average, the resistance signals increased during both hemodialysis sessions and decreased during the interdialytic interval. The increase during dialysis was larger at 8 kHz (∆ 32.6 Ω during session 1 and ∆ 10 Ω during session 2), compared to 160 kHz (∆ 29.5 Ω during session 1 and ∆ 5.1 Ω during session 2). Whereas the resistance at 8 kHz showed a linear time pattern, the evolution of the resistance at 160 kHz was significantly different (p < 0.0001). Measuring bioimpedance semi-continuously and with a multi-frequency current is a major step forward in the understanding of fluid dynamics in hemodialysis patients. This study paves the road towards remote fluid monitoring.

Dormancy and double-activation strategy for construction of high-performance mixed-matrix membranes.

Mixed-matrix membranes (MMMs) have the potential for energy-efficient gas separation by matching the superior mass transfer and anti-plasticization properties of the fillers with processability and scaling up features of the polymers. However, construction of high-performance MMMs has been prohibited due to low filler-loading and the existence of interfacial defects. Here, high MOF-loaded, i.e., 55 wt %, MMMs are developed by a 'dormancy and double-activation' (DDA) strategy. High MOF precursor concentration suppresses crystallization in the membrane casting solution, realizing molecular level mixing of all components. Then, the polymeric matrix was formed with uniform encapsulation of MOF nutrients. Subsequently, double-activation was employed to induce MOF crystallization: the alkali promotes MOFs nucleation to harvest small porous nanocrystals while excessive ligands activate the metal ions to enhance the MOFs conversion. As such, quasi-semi-continuous mass transfer channels can be formed in the MMMs by the connected MOFs nanocrystals to boost the gas permeability. The optimized MMM shows significantly ameliorated CO2 permeability, i.e., 2841 Barrer, five-fold enhancement compared with pristine polymer membrane, with a good CO2 /N2 selectivity of 36. Besides, the nanosized MOFs intensify their interaction with polymer chains, endowing the MMMs with good anti-plasticization behaviour and stability, which advances practical application of MMMs in carbon capture.

Treatment of mixed wastewater by vertical rotating microalgae-bacteria symbiotic biofilm reactor.

A novel vertical rotating microalgae-bacteria symbiotic biofilm reactor was built to treat the mixed wastewater containing municipal and soybean soaking wastewater. The reactor was operated in both sequential batch and semi-continuous modes. Under the sequential batch operation mode, the maximum removal rates for Chemical Oxygen Demand (COD), Total Nitrogen (TN), Total Phosphorus (TP), and Ammonia Nitrogen (NH4+-N) of the mixed wastewater were 95.6 %, 96.1 %, 97.6 %, and 100 %, respectively. During the semi-continuous operation, the water discharge indices decreased gradually and eventually stabilized. At stabilization, the removal rates of COD, TN, and NH4+-N achieved 98 %, 95 %, and 99.9 %, respectively. The maximum biomass productivity of the biofilm was 2.69 g·m-2·d-1. Additionally, the carbohydrate, protein and lipid comprised approximately 22 %, 51 % and 10 % of the dry weight of Chlorella. This study demonstrates the great potential of the microalgae-bacteria symbiotic biofilm system to treat food and domestic wastewater while harvesting microalgal biomass.

Evaluating the potential of semi-continuous itaconic acid fermentation by Aspergillus terreus: operational profile and experiences.

Itaconic acid is an important bio-based chemical. The present study aims to evaluate the applicability of semi-continuous fermentation technique for itaconic acid production by Aspergillus terreus. The fermentation is planned to be connected with bipolar membrane electrodialysis unit for acid recovery. This process allows the reuse of residual glucose from the effluent. Our particular attention was focused on the effect of glucose concentration. Two different glucose supplementation strategies were tested: constant glucose concentration in the refilling medium and adjusted glucose concentration in order to maintain a continuously high - 120 g/L - glucose concentration in the fermentor. The itaconic acid titre, yield and productivity for the 24 h time periods between draining/refilling interventions were investigated. The constantly high glucose concentration in the fermentor resulted in doubled biomass formation. The average itaconic acid titre was 32.9 ± 2.7 g/L. The producing strain formed numerous spores during semi-continuous fermentation that germinated continuously. Yield and volumetric productivity showed a periodic pattern during the procedure.

Optimization of semi-continuous dry anaerobic digestion process and biogas yield of dry yellow corn straw: Based on "gradient anaerobic digestion reactor".

In China, the problem of low biogas yield of traditional biogas projects has become increasingly prominent. This study investigated the effects of different hydraulic retention times (HRTs) on the biogas production efficiency and microbial community under pilot conditions. The results show that the "Gradient anaerobic digestion reactor" can stably carry out semi-continuous dry anaerobic digestion and improve biogas yield. The highest volatile solids (VS) biogas yield (413.73 L/kg VS and 221.61 L CH4/kg VS) and VS degradation rate (48.41%) were observed at an HRT of 25 days. When the HRT was 15 days, the volumetric biogas yield was the highest (2.73 L/L/d, 1.43 L CH4/L/d), but the VS biogas yield and degradation rate were significantly decreased. Microbial analysis showed that HRT significantly affected microbial community. It provides basic data support for the development of a new anaerobic digestion process and the practical application of the straw biogas project in China.

Semi-continuous anaerobic co-digestion of thermal and thermal-alkali processed organic fraction of municipal solid waste: Methane yield, energy analysis, anaerobic microbiome.

The semi-continuous anaerobic co-digestion (AcoD) of thermal and thermal-alkali pretreated organic fraction of municipal solid waste (OFMSW) and sewage sludge (SS) was studied under varying hydraulic retention times (HRT) and organic loading rates (OLR Three semi-continuous digesters were operated under control (non-pre-treated), thermally pretreated (125 °C), and thermal-alkali pretreated (125°C-3g/L NaOH) conditions at variable OLRs at 2.5, 4.0, 5.1, and 7.6 kgVS/m3.d and corresponding HRTs of 30, 20, 15, and 10 days. The 10 and 43% higher methane yield (0.445 m3/kgVS) and 11 and 57% higher VS removal (52%) was achieved for thermal-alkali pretreated digester at 5.1 kgVS/m3.d OLR over thermally pretreated (0.408 m3/kgVS, 45% VS removal) and control digesters (0.310 m3/kgVS, 33% VS removal), respectively. Thermal and thermal-alkali digesters failed on increasing the OLR to 7.6 kgVS/m3.d, whereas the control digester becomes upset at 5.1 kgVS/m3.d OLR. The metagenomic study revealed that Firmicutes, Bacteroidetes, Chloroflexi, Euryarchaeota, Proteobacteria, and Actinobacteria were the predominant bacterial population, whereas Methanosarcina and Methanothrix dominated the archaeal community. Energy balance analysis revealed that thermal alkali pretreatment showed the highest positive energy balance of 114.6 MJ/ton with an energy ratio of 1.25 compared with thermally pretreated (81.5 MJ/ton) and control samples (-46.9 MJ/ton). This work pave the way for scaleup of both thermal and thermal-alkali pre-treatment at 125 °C to realize the techno-economic and energy potential of the process.

Effective Removal of Cu2+ Ions from Aqueous Media Using Poly(acrylamide-co-itaconic acid) Hydrogels in a Semi-Continuous Process.

Adsorption is one of the most crucial processes in water treatment today. It offers a low-cost solution that does not require specialized equipment or state-of-the-art technology while efficiently removing dissolved contaminants, including heavy metals. This process allows for the utilization of natural or artificial adsorbents or a combination of both. In this context, polymeric materials play a fundamental role, as they enable the development of adsorbent materials using biopolymers and synthetic polymers. The latter can be used multiple times and can absorb large amounts of water per gram of polymer. This paper focuses on utilizing adsorption through hydrogels composed of poly(acrylamide-co-itaconic acid) for removing Cu2+ ions dissolved in aqueous media in a semi-continuous process. The synthesized hydrogels were first immersed in 0.1 M NaOH aqueous solutions, enabling OH- ions to enter the gel matrix and incorporate into the polymer surface. Consequently, the copper ions were recovered as Cu(OH)2 on the surface of the hydrogel rather than within it, allowing the solid precipitates to be easily separated by decantation. Remarkably, the hydrogels demonstrated an impressive 98% removal efficiency of the ions from the solution in unstirred conditions at 30 °C within 48 h. A subsequent study involved a serial process, demonstrating the hydrogels' reusability for up to eight cycles while maintaining their Cu2+ ion recovery capacity above 80%. Additionally, these hydrogels showcased their capability to remove Cu2+ ions even from media with ion concentrations below 100 ppm.

A semi-continuous efficient strategy for removing phosphorus and nitrogen from eel aquaculture wastewater using the self-flocculating microalga Desmodesmus sp. PW1.

The phosphorus content in eel aquaculture wastewater exceeds the discharge standard, and the amount of wastewater discharged is substantial. Therefore, there is an urgent need to explore an economical and efficient method of treating aquaculture wastewater. This study explored the use of Desmodesmus sp. PW1, a type of microalgae, to treat eel aquaculture wastewater. By optimizing the conditions, Desmodesmus sp. PW1 achieved a total phosphorus (TP) removal efficiency of 92.3%, as well as total nitrogen (TN) and ammonia nitrogen (NH4+-N) removal efficiency of 99%, using a photoperiod of 24:0, a temperature of 25 °C, and an inoculation amount of 15%. Furthermore, Desmodesmus sp. PW1 demonstrated a high self-flocculating efficiency (>90%) within 100 min of settling, which facilitated biomass recovery. Subsequently, a semi-continuous treatment process mode was established with a sewage renewal rate of 90%. The results showed that after four rounds of sewage renewal operations, the microalgae biomass in the sewage treatment system could be maintained between 160.0 and 220.0 mg/L, and the average removal rate of TP was 0.13 mg/(L * h). The lipid content of algae cells collected in the semi-continuous treatment system for eel aquaculture wastewater was as high as 36.5%, and the biodiesel properties met the biodiesel standards authorized by Europe and the United States. Overall, this study provides an economical and effective strategy for converting wastewater into high-value microalgae products.

The synergetic effect from the combination of different adsorption resins in batch and semi-continuous cultivations of S. Cerevisiae cell factories to produce acetylated Taxanes precursors of the anticancer drug Taxol.

In situ product recovery is an efficient way to intensify bioprocesses as it can perform adsorption of the desired natural products in the cultivation. However, it is common to use only one adsorbent (liquid or solid) to perform the product recovery. For this study, the use of an in situ product recovery method with three combined commercial resins (HP-20, XAD7HP, and HP-2MG) with different chemical properties was performed. A new yeast strain of Saccharomyces cerevisiae was engineered using CRISPR Cas9 (strain EJ2) to deliver heterologous expression of oxygenated acetylated taxanes that are precursors of the anticancer drug Taxol ® (paclitaxel). Microscale cultivations using a definitive screening design (DSD) were set to get the best resin combinations and concentrations to retrieve high taxane titers. Once the best resin treatment was selected by the DSD, semi-continuous cultivation in high throughput microscale was performed to increase the total taxanes yield up to 783 ± 33 mg/L. The best T5α-yl Acetate yield obtained was up to 95 ± 4 mg/L, the highest titer of this compound ever reported by a heterologous expression. It was also observed that by using a combination of the resins in the cultivation, 8 additional uncharacterized taxanes were found in the gas chromatograms compared to the dodecane overlay method. Lastly, the cell-waste reactive oxygen species concentrations from the yeast were 1.5-fold lower in the resin's treatment compared to the control with no adsorbent aid. The possible future implications of this method could be critical for bioprocess intensification, allowing the transition to a semi-continuous flow bioprocess. Further, this new methodology broadens the use of different organisms for natural product synthesis/discovery benefiting from clear bioprocess intensification advantages.

Disease mapping for spatially semi-continuous data by estimating equations with application to dengue control.

Disease mapping is a research field to estimate spatial pattern of disease risks so that areas with elevated risk levels can be identified. The motivation of this article is from a study of dengue fever infection, which causes seasonal epidemics in almost every summer in Taiwan. For analysis of zero-inflated data with spatial correlation and covariates, current methods would either cause a computational burden or miss associations between zero and non-zero responses. In this article, we develop estimating equations for a mixture regression model that accommodates spatial dependence and zero inflation for study of disease propagation. Asymptotic properties for the proposed estimates are established. A simulation study is conducted to evaluate performance of the mixture estimating equations; and a dengue dataset from southern Taiwan is used to illustrate the proposed method.

From Batch to the Semi-Continuous Flow Hydrogenation of pNB, pNZ-Protected Meropenem.

Meropenem is currently the most common carbapenem in clinical applications. Industrially, the final synthetic step is characterized by a heterogeneous catalytic hydrogenation in batch mode with hydrogen and Pd/C. The required high-quality standard is very difficult to meet and specific conditions are required to remove both protecting groups [i.e., p-nitrobenzyl (pNB) and p-nitrobenzyloxycarbonyl (pNZ)] simultaneously. The three-phase gas-liquid-solid system makes this step difficult and unsafe. The introduction of new technologies for small-molecule synthesis in recent years has opened up new landscapes in process chemistry. In this context, we have investigated meropenem hydrogenolysis using microwave (MW)-assisted flow chemistry for use as a new technology with industrial prospects. The reaction parameters (catalyst amount, T, P, residence time, flow rate) in the move from the batch process to semi-continuous flow were investigated under mild conditions to determine their influence on the reaction rate. The optimization of the residence time (840 s) and the number of cycles (4) allowed us to develop a novel protocol that halves the reaction time compared to batch production (14 min vs. 30 min) while maintaining the same product quality. The increase in productivity using this semi-continuous flow technique compensates for the slightly lower yield (70% vs. 74%) obtained in batch mode.

Dynamics of a within-host drug resistance model with impulsive state feedback control.

Bacterial resistance poses a major hazard to human health, and is caused by the misuse and overuse of antibiotics. Thus, it is imperative to investigate the optimal dosing strategy to improve the treatment effect. In this study, a mathematical model of antibiotic-induced resistance is presented to improve the antibiotic effectiveness. First, conditions for the global asymptotical stability of the equilibrium without pulsed effect are given according to the Poincaré-Bendixson Theorem. Second, a mathematical model of the dosing strategy with impulsive state feedback control is also formulated to reduce drug resistance to an acceptable level. The existence and stability of the order-1 periodic solution of the system are discussed to obtain the optimal control of antibiotics. Finally, our conclusions are confirmed by means of numerical simulations.

Efficient production of hyaluronic acid by Streptococcus zooepidemicus using two-stage semi-continuous fermentation.

Hyaluronic acid is a kind of mucopolysaccharide that has wide applications in cosmetics, health food, and orthopedics. Using Streptococcus zooepidemicus ATCC 39920 as parent, a beneficial mutant SZ07 was obtained by UV mutagenesis, giving 1.42 g/L hyaluronic acid in shake flasks. To enhance the efficiency of hyaluronic acid production, a semi-continuous fermentation process consisted of two-stage 3-L bioreactors was developed, in which 1.01 g/L/h productivity and 14.60 g/L hyaluronic acid were obtained. To further enhance the titer of hyaluronic acid, recombinant hyaluronidase SzHYal was added into 2nd stage bioreactor at 6 h to reduce the viscosity of broth. The highest hyaluronic acid titer of 29.38 g/L was achieved with a productivity of 1.13 g/L/h at 300 U/L SzHYal after 24 h. This newly developed semi-continuous fermentation process provides a promising strategy for the industrial production of hyaluronic acid and related polysaccharides.

Biochemical hydrogen potential assay for predicting the patterns of the kinetics of semi-continuous dark fermentation.

The performance and kinetics response of thermophilic semi-continuous dark fermentation (DF) of simulated complex carbohydrate-rich waste was investigated at various hydraulic retention times (HRT) (2, 2.5, and 3 d) and compared with data obtained from biochemical hydrogen potential assay (BHP). A culture of Thermoanaerobacterium thermosaccharolyticum was used as the inoculum and dominated both in BHP and semi-continuous reactor. Both the modified Gompertz and first-order models described the DF kinetics well (R2 = 0.97-1.00). HRT of 2.5 d was found to be optimal in terms of maximum hydrogen production rate and hydrogen potential, which were 3.97 and 1.26 times higher, respectively, than in BHP. The hydrolysis constant was highest at HRT of 3 d and was closest to the value obtained in the BHP. Overall, BHP has been shown to be a useful tool for predicting H2 potential and the hydrolysis constant, while the maximum H2 production rate is greatly underestimated.