This work assessed the performance of a pilot-scale cascade anaerobic digestion (AD) system when treating mixed municipal wastewater treatment sludges. The cascade system was compared with a conventional continuous stirred tank reactor (CSTR) digester (control) in terms of process performance, stability, and digestate quality. The results showed that the cascade system achieved higher volatile solids removal (VSR) efficiencies (28-48%) than that of the reference (25-41%) when operated at the same solids residence time (SRT) in the range of 11-15 days. When the SRT of the cascade system was reduced to 8 days the VSR (32-36%) was only slightly less than that of the reference digester that was operated at a 15-day SRT (39-43%). Specific hydrolysis rates in the first stage of the cascade system were 66-152% higher than those of the reference. Additionally, the cascade system exhibited relatively stable effluent concentrations of volatile fatty acids (VFAs: 100-120 mg/l), while the corresponding concentrations in the control effluent demonstrated greater fluctuations (100-160 mg/l). The cascade system\'s effluent pH and VFA/alkalinity ratios were consistently maintained within the optimal range. During a dynamic test when the feed total solids concentration was doubled, total VFA concentrations (85-120 mg/l) in the cascade system were noticeably less than those (100-170 mg/l) of the control, while the pH and VFA/alkalinity levels remained in a stable range. The cascade system achieved higher total solids (TS) content in the dewatered digestate (19.4-26.8%) than the control (17.4-22.1%), and E. coli log reductions (2.0-4.1 log MPN/g TS) were considerably higher (p < 0.05) than those in the control (1.3-2.9 log MPN/g TS). Overall, operating multiple CSTRs in cascade mode at typical SRTs and mixed sludge ratios enhanced the performance, stability digesters, and digestate quality of AD. PRACTITIONER POINTS: Enhanced digestion of mixed sludge digestion with cascade system. Increased hydrolysis rates in the cascade system compared to a reference CSTR. More stable conditions for methanogen growth at both steady and dynamic states. Improved dewaterability and E. coli reduction of digestate from the cascade system.

Liquid-liquid reactions play a significant role in organic synthesis. However, control of the phase interface between incompatible two-phase liquids remains challenging. Moreover, separating liquid acid, base and oxidants from the reactor takes a long time and high cost. To address these issues, we draw inspiration from the structure and function of cells in living organisms and develop a biomimetic 3D-printed cellular reactor. The cellular reactor houses an aqueous phase containing the catalyst or oxidant while immersed in the organic phase reactant. This setup controls the distribution of the phase interface within the organic phase and increases the interface area by 2.3 times. Notably, the cellular reactor and the aqueous phase are removed from the organic phase upon completing the reaction, eliminating additional separation steps and preventing direct contact between the reactor and acidic, alkaline, or oxidizing substances. Furthermore, the cellular reactor offers the advantages of digital design feasibility and cost-effective manufacturing.

Mesenchymal stem and stromal cells (MSCs) hold potential to treat a broad range of clinical indications, but clinical translation has been limited to date due in part to challenges with batch-to-batch reproducibility of potential critical quality attributes (pCQAs) that can predict potency/efficacy. Here, we designed and implemented a microcarrier-microbioreactor approach to cell therapy manufacturing, specific to anchorage-dependent cells such as MSCs. We sought to assess whether increased control of the biochemical and biophysical environment had the potential to create product with consistent presentation and elevated expression of pCQAs relative to established manufacturing approaches in tissue culture polystyrene (TCPS) flasks. First, we evaluated total cell yield harvested from dissolvable, gelatin microcarriers within a microbioreactor cassette (Mobius Breez) or a flask control with matched initial cell seeding density and culture duration. Next, we identified 24 genes implicated in a therapeutic role for a specific motivating indication, acute respiratory distress syndrome (ARDS); expression of these genes served as our pCQAs for initial in vitro evaluation of product potency. We evaluated mRNA expression for three distinct donors to assess inter-donor repeatability, as well as for one donor in three distinct batches to assess within-donor, inter-batch variability. Finally, we assessed gene expression at the protein level for a subset of the panel to confirm successful translation. Our results indicated that MSCs expanded with this microcarrier-microbioreactor approach exhibited reasonable donor-to-donor repeatability and reliable batch-to-batch reproducibility of pCQAs. Interestingly, the baseline conditions of this microcarrier-microbioreactor approach also significantly improved expression of several key pCQAs at the gene and protein expression levels and reduced total media consumption relative to TCPS culture. This proof-of-concept study illustrates key benefits of this approach to therapeutic cell process development for MSCs and other anchorage-dependent cells that are candidates for cell therapies.

BACKGROUND: Stem cell-derived therapies hold the potential for treatment of regenerative clinical indications. Static culture has a limited ability to scale up thus restricting its use. Suspension culturing can be used to produce target cells in large quantities, but also presents challenges related to stress and aggregation stability.
METHODS: Utilizing a design of experiments (DoE) approach in vertical wheel bioreactors, we evaluated media additives that have versatile properties. The additives evaluated are Heparin sodium salt (HS), polyethylene glycol (PEG), poly (vinyl alcohol) (PVA), Pluronic F68 and dextran sulfate (DS). Multiple response variables were chosen to assess cell growth, pluripotency maintenance and aggregate stability in response to the additive inputs, and mathematical models were generated and tuned for maximal predictive power.
RESULTS: Expansion of iPSCs using 100 ml vertical wheel bioreactor assay for 4 days on 19 different media combinations resulted in models that can optimize pluripotency, stability, and expansion. The expansion optimization resulted in the combination of PA, PVA and PEG with E8. This mixture resulted in an expansion doubling time that was 40% shorter than that of E8 alone. Pluripotency optimizer highlighted the importance of adding 1% PEG to the E8 medium. Aggregate stability optimization that minimizes aggregate fusion in 3D culture indicated that the interaction of both Heparin and PEG can limit aggregation as well as increase the maintenance capacity and expansion of hiPSCs, suggesting that controlling fusion is a critical parameter for expansion and maintenance. Validation of optimized solution on two cell lines in bioreactors with decreased speed of 40 RPM, showed consistency and prolonged control over aggregates that have high frequency of pluripotency markers of OCT4 and SOX2 (> 90%). A doubling time of around 1-1.4 days was maintained after passaging as clumps in the optimized medium. Controlling aggregate fusion allowed for a decrease in bioreactor speed and therefore shear stress exerted on the cells in a large-scale expansion.
CONCLUSIONS: This study resulted in a control of aggregate size within suspension cultures, while informing about concomitant state control of the iPSC state. Wider application of this approach can address media optimization complexity and bioreactor scale-up challenges.

This study evaluated the treatment efficiency of two selected fillers and their combination for improving the water quality of aquaculture wastewater using a packed bed biofilm reactor (PBBR) under various process conditions. The fillers used were nanosheet (NS), activated carbon (AC), and a combination of both. The results indicated that the use of combined fillers and the hydraulic retention time (HRT) of 4 h significantly enhanced water quality in the PBBR. The removal rates of chemical oxygen demand, NO2-─N, total suspended solids（TSS）, and chlorophyll a were 63.55%, 74.25%, 62.75%, and 92.85%, respectively. The microbiota analysis revealed that the presence of NS increased the abundance of microbial phyla associated with nitrogen removal, such as Nitrospirae and Proteobacteria. The difference between the M1 and M2 communities was minimal. Additionally, the microbiota in different PBBR samples displayed similar preferences for carbon sources, and carbohydrates and amino acids were the most commonly utilized carbon sources by microbiota. These results indicated that the combination of NS and AC fillers in a PBBR effectively enhanced the treatment efficiency of aquaculture wastewater when operated at an HRT of 4 h. The findings provide valuable insights into optimizing the design of aquaculture wastewater treatment systems.

Rice wine, well known for its unique flavor, rich nutritional value, and health benefits, has potential for extensive market development. Rhizopus and Aspergillus are among several microorganisms used in rice wine brewing and are crucial for determining rice wine quality. The strains were isolated via Rose Bengal and starch as a combined separation medium, followed by oenological property and sensory evaluation screening. The strain exhibiting the best performance can be screened using the traditional rice wine Qu. The strains YM-8, YM-10, and YM-16, which exhibited strong saccharification and fermentation performance along with good flavor and taste, were obtained from traditional rice wine Qu. Based on ITS genetic sequence analysis, the YM-8, YM-10, and YM-16 strains were identified as Rhizopus microsporus, Rhizopus arrhizus, and Aspergillus oryzae. The optimum growth temperature of each of the three strains was 30°C, 32°C, and 30°C, and the optimum initial pH was 6.0, 6.5, and 6.5, respectively. The activities of α-amylase, glucoamylase, and protease of YM-16 were highest at 220.23±1.88, 1,269.04±30.32, and 175.16±1.81 U/g, respectively. The amino acid content of rice wine fermented in a 20-L bioreactor with the three mold strains was higher than that of the control group, except for arginine, which was significantly lower than that of the control group. The total amino acid content and the total content of each type of amino acid were ranked as YM-16 > YM-8 > YM-10 > control group, and the amino acid content varied greatly among the strains. The control group had a higher content, whereas YM-8 and YM-16 had lower contents of volatile aroma components than the control group and had the basic flavor substances needed for rice wine, which is conducive to the formation of rice wine aroma. This selected strain, YM-16, has strong saccharification and fermentation ability, is a rich enzyme system, and improves the flavor of rice wine, thereby demonstrating its suitability as a production strain for brewing.

Colistin remains an important antibiotic for the therapeutic management of drug-resistant Klebsiella pneumoniae. Despite the numerous reports of colistin resistance in clinical strains, it remains unclear exactly when and how different mutational events arise resulting in reduced colistin susceptibility. Using a bioreactor model of infection, we modelled the emergence of colistin resistance in a susceptible isolate of K. pneumoniae. Genotypic, phenotypic and mathematical analyses of the antibiotic-challenged and un-challenged population indicates that after an initial decline, the population recovers within 24 h due to a small number of \"founder cells\" which have single point mutations mainly in the regulatory genes encoding crrB and pmrB that when mutated results in up to 100-fold reduction in colistin susceptibility. Our work underlines the rapid development of colistin resistance during treatment or exposure of susceptible K. pneumoniae infections having implications for the use of cationic antimicrobial peptides as a monotherapy.

The use of animal models is still widespread in science but there is a movement away from this manner of experimentation. One option approved by the FDA for human-like studies is the hollow fiber bioreactor (HFS). HFSs are highly controllable, self-contained systems that allow for the modeling of individual tissues and disease phenotypes. Oxygen, drug concentration & half-life, and immune cell invasion are all scalable to human and veterinary conditions using a HFS. There are drawbacks to the systems including cost and contamination so the use of these systems must be carefully managed.With these limitations in mind, the scope of the technology is great. Antimicrobial susceptibility testing (AST) is possible with greater accuracy and clinical validity than classical in vitro techniques making minimal inhibitory concentration (MIC) data generated on the bench more translatable to the clinic.In this chapter, we will outline the background of the HFS and some typical uses.

Therapeutic monoclonal antibody (mAb) development and the processes for manufacturing drug substance have evolved since the first approval of the mAb in 1986. As the past is often the prologue to the future, the history of these technologies has been classified here into three eras, leading to speculation about what the next era may hold with regard to development and manufacturing strategies, as well as the potential impacts to patients. The substantial increase in production culture titers and bioreactor production volumes and the availability of large-scale contract manufacturing facilities could translate into improved global access for these therapies and an expansion of indications for therapeutic antibodies.

DNA serves as a model system in polymer physics due to its ability to be obtained as a uniform polymer with controllable topology and nonequilibrium behavior. Currently, a major obstacle in the widespread adoption of DNA is obtaining it on a scale and cost basis that accommodates bulk rheology and high-throughput screening. To address this, recent advancements in bioreactor-based plasmid DNA production is coupled with anion exchange chromatography producing a unified approach to generating gram-scale quantities of monodisperse DNA. With this method, 1.1 grams of DNA is obtained per batch to generate solutions with concentrations up to 116 mg mL-1. This solution of uniform supercoiled and relaxed circular plasmid DNA, is roughly 69 times greater than the overlap concentration. The utility of this method is demonstrated by performing bulk rheology measurements at sample volumes up to 1 mL on DNA of different lengths, topologies, and concentrations. The measured elastic moduli are orders of magnitude larger than those previously reported for DNA and allowed for the construction of a time-concentration superposition curve that spans 12 decades of frequency. Ultimately, these results can provide important insights into the dynamics of ring polymers and the nature of highly condensed DNA dynamics.