Transcriptomic profiling of human mesenchymal stem cells using a pulsed electromagnetic-wave motion bioreactor system for enhanced osteogenic commitment and therapeutic potentials.

Traditional bioreactor systems involve the use of three-dimensional (3D) scaffolds or stem cell aggregates, limiting the accessibility to the production of cell-secreted biomolecules. Herein, we present the use a pulse electromagnetic fields (pEMFs)-assisted wave-motion bioreactor system for the dynamic and scalable culture of human bone marrow-derived mesenchymal stem cells (hBMSCs) with enhanced the secretion of various soluble factors with massive therapeutic potential. The present study investigated the influence of dynamic pEMF (D-pEMF) on the kinetic of hBMSCs. A 30-min exposure of pEMF (10V-1Hz, 5.82 G) with 35 oscillations per minute (OPM) rocking speed can induce the proliferation (1 × 105 â†’ 4.5 × 105) of hBMSCs than static culture. Furthermore, the culture of hBMSCs in osteo-induction media revealed a greater enhancement of osteogenic transcription factors under the D-pEMF condition, suggesting that D-pEMF addition significantly boosted hBMSCs osteogenesis. Additionally, the RNA sequencing data revealed a significant shift in various osteogenic and signaling genes in the D-pEMF group, further suggesting their osteogenic capabilities. In this research, we demonstrated that the combined effect of wave and pEMF stimulation on hBMSCs allows rapid proliferation and induces osteogenic properties in the cells. Moreover, our study revealed that D-pEMF stimuli also induce ROS-scavenging properties in the cultured cells. This study also revealed a bioactive and cost-effective approach that enables the use of cells without using any expensive materials and avoids the possible risks associated with them post-implantation.

Evaluation of the shear stability of aerobic granular sludge from a pilot-scale membrane bioreactor: Establishment of a quantitative method.

This work established a quantitative method to access the shear stability of aerobic granular sludge (AGS) and validated its feasibility by using the mature AGS from a pilot-scale (50 tons/day) membrane bioreactor (MBR) for treating real municipal wastewater. The results showed that the changing rate (ΔS) of the peak area (S) of granule size distribution (GSD) exhibited an exponential relationship (R2≥0.76) with the shear time (y=a-b·cx), which was a suitable indicative index to reflect the shear stability of different AGS samples. The limiting granule size (LGS) was defined and proposed to characterize the equilibrium size for AGS after being sheared for a period of time, whose value in terms of Dv50 showed high correlation (R2=0.92) with the parameter a. The free Ca2+ (28.44-34.21 mg/L) in the influent specifically interacted with polysaccharides (PS) in the granule's extracellular polymeric substance (EPS) as a nucleation site, thereby inducing the formation of Ca precipitation to enhance its Young's modulus, while Ca2+ primarily interacted with PS in soluble metabolic product (SMP) during the initial granulation process. Furthermore, the Young's modulus significantly affected the parameter a related to shear stability (R2=0.99). Since the parameter a was more closely related (R2=1.00) to ΔS than that of the parameter b or c, the excellent correlation (R2=0.99) between the parameter a and the wet density further verified the feasibility of this method.

Sustainable control of microplastics in wastewater using the electrochemically enhanced living membrane bioreactor.

Wastewater treatment plant (WWTP) discharges are major contributors to the release of microplastics (MPs) into the environment. This research work aimed to assess the performance of the novel living membrane bioreactor (LMBR), which utilizes a biological layer as a membrane filter for the removal of polyethylene (PE) MPs from wastewater. The impact of an intermittently applied low current density (0.5 mA/cm2) on the reduction of MPs in the electrochemically enhanced LMBR (e-LMBR) has also been examined. The reactors were also compared to a conventional membrane bioreactor (MBR) and an electro-MBR (e-MBR). 1H nuclear magnetic resonance spectroscopy (1H NMR) was implemented for the MPs detection and quantification in terms of mass per volume of sample. The LMBR and MBR achieved comparable mean PE MPs reduction at 95% and 96%, respectively. The MPs mass reduction in the e-LMBR slightly decreased by 2% compared to that achieved in the LMBR. This potentially indicated the partial breakdown of the MPs due to electrochemical processes. Decreasing and inconsistent NH4-N and PO4-P removal efficiencies were observed over time due to the addition of PE MPs in the MBR and LMBR. In contrast, the integration of electric field in the e-MBR and e-LMBR resulted in consistently high values of conventional contaminant removals of COD (99.72-99.77 %), NH4-N (97.96-98.67%), and PO4-P (98.44-100.00%), despite the MPs accumulation. Integrating electrochemical processes in the e-LMBR led to the development of a stable living membrane (LM) layer, as manifested in the consistently low effluent turbidity 0.49 ± 0.33 NTU. Despite the increasing MPs concentration in the mixed liquor, applying electrochemical processes reduced the fouling rates in the e-LMBR. The e-LMBR achieved comparable efficiencies in contaminant reductions as those observed in the e-MBR, while using a low-cost membrane material.

Tonsil explants as a human in vitro model to study vaccine responses.

Introduction: Vaccination is one of the most effective infection prevention strategies. Viruses with high mutation rates -such as influenza- escape vaccine-induced immunity and represent significant challenges to vaccine design. Influenza vaccine strain selection is based on circulating strains and immunogenicity testing in animal models with limited predictive outcomes for vaccine effectiveness in humans. Methods: We developed a human in vitro vaccination model using human tonsil tissue explants cultured in 3D perfusion bioreactors to be utilized as a platform to test and improve vaccines. Results: Tonsils cultured in bioreactors showed higher viability, metabolic activity, and more robust immune responses than those in static cultures. The in vitro vaccination system responded to various premanufactured vaccines, protein antigens, and antigen combinations. In particular, a multivalent in vitro immunization with three phylogenetically distant H3N2 influenza strains showed evidence for broader B cell activation and induced higher antibody cross-reactivity than combinations with more related strains. Moreover, we demonstrate the capacity of our in vitro model to generate de novo humoral immune responses to a model antigen. Discussion: Perfusion-cultured tonsil tissue may be a valuable human in vitro model for immunology research with potential application in vaccine candidate selection.

Pre-vascularized porous gelatin-coated ß-tricalcium phosphate scaffolds for bone regeneration: an in vivo and in vitro investigation.

Vascularization is vital in bone tissue engineering, supporting development, remodeling, and regeneration. Lack of vascularity leads to cell death, necessitating vascularization strategies. Angiogenesis, forming new blood vessels, provides crucial nutrients and oxygen. Pre-vascularized gelatin-coated ß-tricalcium phosphate (G/ß-TCP) scaffolds show promise in bone regeneration and vascularization. Our study evaluates G/ß-TCP scaffolds' osteogenic and angiogenic potential in vitro and a canine model with vascular anastomosis. Channel-shaped G/ß-TCP scaffolds were fabricated using foam casting and sintering of a calcium phosphate/silica slurry-coated polyurethane foam, then coated with cross-linked gelatin. Buccal fat pad-derived stem cells (BFPdSCs) were seeded onto scaffolds and assessed over time for adhesion, proliferation, and osteogenic capacity using scanning electron microscopy (SEM), 4,6-diamidino-2-phenylindole (DAPI) staining, Alamar blue, and alkaline phosphatase (ALP) assays. Scaffolds were implanted in a canine model to evaluate osteogenesis and angiogenesis by histology and CT scans at 12 wk. Our studies showed preliminary results for G/ß-TCP scaffolds supporting angiogenesis and bone regeneration. In vitro analyses demonstrated excellent proliferation/viability, with BFPdSCs adhering and increasing on the scaffolds. ALP activity and protein levels increased, indicating osteogenic differentiation. Examination of tissue samples revealed granulation tissue with a well-developed vascular network, indicating successful angiogenesis and osteogenesis was further confirmed by a CT scan. In vivo, histology revealed scaffold resorption. However, scaffold placement beneath muscle tissue-restricted bone regeneration. Further optimization is needed for bone regeneration applications.

Comparative evaluation of the extracellular production of a polyethylene terephthalate degrading cutinase by Corynebacterium glutamicum and leaky Escherichia coli in batch and fed-batch processes.

BACKGROUND: With a growing global population, the generation of plastic waste and the depletion of fossil resources are major concerns that need to be addressed by developing sustainable and efficient plastic recycling methods. Biocatalytic recycling is emerging as a promising ecological alternative to conventional processes, particularly in the recycling of polyethylene terephthalate (PET). However, cost-effective production of the involved biocatalyst is essential for the transition of enzymatic PET recycling to a widely used industrial technology. Extracellular enzyme production using established organisms such as Escherichia coli or Corynebacterium glutamicum offers a promising way to reduce downstream processing costs. RESULTS: In this study, we compared extracellular recombinant protein production by classical secretion in C. glutamicum and by membrane leakage in E. coli. A superior extracellular release of the cutinase ICCGDAQI was observed with E. coli in batch and fed-batch processes on a litre-scale. This phenomenon in E. coli, in the absence of a signal peptide, might be associated with membrane-destabilizing catalytic properties of the expressed cutinase. Optimisations regarding induction, expression temperature and duration as well as carbon source significantly enhanced extracellular cutinase activity. In particular, in fed-batch cultivation of E. coli at 30 °C with lactose as carbon source and inducer, a remarkable extracellular activity (137 U mL-1) and cutinase titre (660 mg L-1) were achieved after 48 h. Literature values obtained with other secretory organisms, such as Bacillus subtilis or Komagataella phaffii were clearly outperformed. The extracellular ICCGDAQI produced showed high efficacy in the hydrolysis of PET textile fibres, either chromatographically purified or unpurified as culture supernatant. In less than 18 h, 10 g L-1 substrate was hydrolysed using supernatant containing 3 mg cutinase ICCGDAQI at 70 °C, pH 9 with terephthalic acid yields of up to 97.8%. CONCLUSION: Extracellular production can reduce the cost of recombinant proteins by simplifying downstream processing. In the case of the PET-hydrolysing cutinase ICCGDAQI, it was even possible to avoid chromatographic purification and still achieve efficient PET hydrolysis. With such production approaches and their further optimisation, enzymatic recycling of PET can contribute to a more efficient and environmentally friendly solution to the industrial recycling of plastics in the future.

Manufacturing of the highly active thermophile PETases PHL7 and PHL7mut3 using Escherichia coli.

BACKGROUND: The global plastic waste crisis requires combined recycling strategies. One approach, enzymatic degradation of PET waste into monomers, followed by re-polymerization, offers a circular economy solution. However, challenges remain in producing sufficient amounts of highly active PET-degrading enzymes without costly downstream processes. RESULTS: Using the growth-decoupled enGenes eX-press V2 E. coli strain, pH, induction strength and feed rate were varied in a factorial-based optimization approach, to find the best-suited production conditions for the PHL7 enzyme. This led to a 40% increase in activity of the fermentation supernatant. Optimization of the expression construct resulted in a further 4-fold activity gain. Finally, the identified improvements were applied to the production of the more active and temperature stable enzyme variant, PHL7mut3. The unpurified fermentation supernatant of the PHL7mut3 fermentation was able to completely degrade our PET film sample after 16 h of incubation at 70 °C at an enzyme loading of only 0.32 mg enzyme per g of PET. CONCLUSIONS: In this research, we present an optimized process for the extracellular production of thermophile and highly active PETases PHL7 and PHL7mut3, eliminating the need for costly purification steps. These advancements support large-scale enzymatic recycling, contributing to solving the global plastic waste crisis.

Pilot scale production of high-content mycoprotein using Rhizopus microsporus var. oligosporus by submerged fermentation and agro-industrial by-products.

While mycoprotein has gained traction as a human food source, its potential as a nutrient for animals remains largely unexplored. The mycoprotein-producing Rhizopus microsporus var. oligosporus, a fungus traditionally used for human food in Indonesia, is promising. It could revolutionise animal nutrition once it is Generally Recognized as Safe (GRAS) and is a biosafety level 1 (BSL1) organism. To enhance sustainably, we propose using sugar cane molasses (SM) and corn steep liquor (CSL) as nutrient sources. Also, we investigated the growth of R. microsporus var. oligosporus in five 14 L external-loop airlift bioreactors using CSL as the sole nutrient source. After 96 h of fermentation, at 25 °C and 0.5 vvm, the mycelium produced had an average biomass yield of 38.34 g L-1, with 70.18 % (m v-1) crude protein (mycoprotein). This bioprocess, which is scalable and economically viable, produces high amounts of mycoprotein for animal feed using CSL, a cost-effective agro-industrial by-product, providing a practical solution to the growing demand for animal protein.

Quantitative investigation of the effect of mechanical and geometrical factors of a laboratory-scale bioreactor using a vibrating agitator on mammalian cell culture indices.

Mammalian cell cultures in laboratories are performed in static and dynamic methods, and cell growth indices are higher in dynamic mode. In this study, a lab-scale stirred bioreactor using a vibrating disc and a suitable setup has been introduced for dynamic cell culture, which creates proper mixing at low shear stress. 15 experiments have been done by Raji cell in batch mode using Box-Behnken design to quantitatively investigate the effect of mechanical and geometrical factors of this bioreactor on cell culture indices. Three structural factors, including disc diameter, vibration amplitude, and the height of the disc placement have been selected as the main factors. Three cell growth indices including the specific growth rate, the maximum cell concentration, and productivity have been considered as biological responses. Resulting models predict the value of each index under different settings of the factors with good accuracy. Results show that the disc diameter has the greatest effect among the investigated factors. Also, the specific growth rate, the natural logarithm of the maximum cell concentration, and productivity are about 0.033 (1/h), 13.2, and 5133 (cells/hmL), respectively by using a 25 (mm) disc with a vibration amplitude of 2.5 up to 3 (mm), and a placement height of 40 up to 60 (mm).

Growth of sulfate-reducing Desulfobacterota and Bacillota at periodic oxygen stress of 50% air-O2 saturation.

BACKGROUND: Sulfate-reducing bacteria (SRB) are frequently encountered in anoxic-to-oxic transition zones, where they are transiently exposed to microoxic or even oxic conditions on a regular basis. This can be marine tidal sediments, microbial mats, and freshwater wetlands like peatlands. In the latter, a cryptic but highly active sulfur cycle supports their anaerobic activity. Here, we aimed for a better understanding of how SRB responds to periodically fluctuating redox regimes. RESULTS: To mimic these fluctuating redox conditions, a bioreactor was inoculated with peat soil supporting cryptic sulfur cycling and consecutively exposed to oxic (one week) and anoxic (four weeks) phases over a period of > 200 days. SRB affiliated to the genus Desulfosporosinus (Bacillota) and the families Syntrophobacteraceae, Desulfomonilaceae, Desulfocapsaceae, and Desulfovibrionaceae (Desulfobacterota) successively established growing populations (up to 2.9% relative abundance) despite weekly periods of oxygen exposures at 133 µM (50% air saturation). Adaptation mechanisms were analyzed by genome-centric metatranscriptomics. Despite a global drop in gene expression during oxic phases, the perpetuation of gene expression for energy metabolism was observed for all SRBs. The transcriptional response pattern for oxygen resistance was differentiated across individual SRBs, indicating different adaptation strategies. Most SRB transcribed differing sets of genes for oxygen consumption, reactive oxygen species detoxification, and repair of oxidized proteins as a response to the periodical redox switch from anoxic to oxic conditions. Noteworthy, a Desulfosporosinus, a Desulfovibrionaceaea, and a Desulfocapsaceaea representative maintained high transcript levels of genes encoding oxygen defense proteins even under anoxic conditions, while representing dominant SRB populations after half a year of bioreactor operation. CONCLUSIONS: In situ-relevant peatland SRB established large populations despite periodic one-week oxygen levels that are one order of magnitude higher than known to be tolerated by pure cultures of SRB. The observed decrease in gene expression regulation may be key to withstand periodically occurring changes in redox regimes in these otherwise strictly anaerobic microorganisms. Our study provides important insights into the stress response of SRB that drives sulfur cycling at oxic-anoxic interphases. Video Abstract.

The proliferation and differentiation of skeletal muscle stem cells are enhanced in a bioreactor.

Skeletal muscle (SKM) is the largest organ in mammalian body and it can repair damages by using the residential myogenic stem cells (MuSC), but this repairing capacity reduces with age and in some genetic muscular dystrophy. Under these circumstances, artificial amplification of autologous MuSC in vitro might be necessary to repair the damaged SKM. The amplification of MuSC is highly dependent on myogenic signals, such as sonic hedgehog (Shh), Wnt3a, and fibroblast growth factors, so formulating an optimum myogenic kit composed of specific myogenic signals might increase the proliferation and differentiation of MuSC efficiently. In this study, various myogenic signals have been tested on C2C12 myoblasts and primary MuSC, and a myogenic kit consists of insulin, lithium chloride, T3, and retinoic acid has been formulated, and we found it significantly increased the fusion index and MHC expression level of both C2C12 and MuSC myotubes. A novel bioreactor providing cyclic stretching (CS) and electrical stimulation (ES) has been fabricated to enhance the myogenic differentiation of both C2C12 and MuSC. We further found that coating the bioreactor substratum with collagen gave the best effect on proliferation and differentiation of MuSC. Furthermore, combining the collagen coating and physical stimuli (CS + ES) in the bioreactor can generate more proliferative primary MuSC cells. Our results have demonstrated that the combination of myogenic kit and bioreactor can provide environment for efficient MuSC proliferation and differentiation. These MuSC and mature myotubes amplified in the bioreactor might be useful for clinical grafting into damaged SKM in the future.

Alternative Method for Obtaining Human-Induced Pluripotent Stem Cell Lines and Three-Dimensional Growth: A Simplified, Passage-Free Approach that Minimizes Labor.

Induced pluripotent stem cells (iPSCs) hold significant promise for numerous applications in regenerative medicine, disease modeling, and drug discovery. However, the conventional workflow for iPSC generation, with cells grown under two-dimensional conditions, presents several challenges, including the need for specialized scientific skills such as morphologically assessing and picking colonies and removing differentiated cells during the establishment phase. Furthermore, maintaining established iPSCs in three-dimensional culture systems, while offering scalability, necessitates an enzymatic dissociation step for their further growth in a complex and time-consuming protocol. In this study, we introduce a novel approach to address these challenges by reprogramming somatic cells grown under three-dimensional conditions as spheres using a bioreactor, thereby eliminating the need for two-dimensional culture and colony picking. The iPSCs generated in this study were maintained under three-dimensional conditions simply by transferring spheres to the next bioreactor, without the need for an enzymatic dissociation step. This streamlined method simplifies the workflow, reduces technical variability and labor, and paves the way for future advancements in iPSC research and its wider applications. Key features • Establishment of induced pluripotent stem cells in a three-dimensional environment. • Maintenance and cryopreservation of iPSCs without the need for a dissociation step.

A dynamically loaded ex vivo model to study neocartilage and integration in human cartilage repair.

Articular cartilage injuries in the knee can lead to post-traumatic osteoarthritis if untreated, causing debilitating problems later in life. Standard surgical treatments fail to ensure long lasting repair of damaged cartilage, often resulting in fibrotic tissue. While there is a vast amount of research into cartilage regeneration, integrating engineered implants with cartilage remains a challenge. As cartilage is a load bearing tissue, it is imperative to evaluate tissue repair strategies and their ability to integrate under mechanical loading. This work established a dynamically loaded ex vivo model of cartilage repair using human cartilage explants. The model was used to assess the efficacy of a stem cell therapy delivered in a bioadhesive hydrogel comprised of photocrosslinkable gelatin methacryloyl (GelMA) and microbial transglutaminase to repair the model defect. Extensive neocartilage production and integration were observed via histology and immunohistochemistry after 28 days chondrogenic culture. Analysis of culture media allowed monitoring of glycosaminoglycan and type II collagen production over time. A mechanical assessment of integration via a push out test showed a 15-fold increase in push out strength over the culture duration. The model was successful in exhibiting robust chondrogenesis with transglutaminase or without, and under both culture conditions. The work also highlights several limitations of ex vivo models and challenges of working with bioreactors that must be overcome to increase their utility. This ex vivo model has the potential to delay the need for costly pre-clinical studies and provide a more nuanced assessment of cartilage repair strategies than is possible in vivo.

Triple enzymatic cascade reaction to produce hydroxytyrosol acetate from olive leaves using integrated membrane bioreactors.

An integrated system of three membrane bioreactors (MBRs) has been developed that cascades three different enzymatic reactions. The integrated system was applied to produce hydroxytyrosol acetate from oleuropein extracted from olive leaves. Different reactor configurations for each reaction were tested and individually optimized to select the MBR to ensure high conversion and continuous production of oleuropein aglycone (OA), hydroxytyrosol (HY) and hydroxytyrosol acetate (HA). Based on this study, the most performing configuration of the integrated system was identified. In the first reaction, oleuropein was converted to OA using a biocatalytic membrane reactor (BMR) with immobilized ß-glucosidase in polymeric membranes (conversion 98%). The OA was then fed to another BMR, where it was converted to HY (conversion: 70%) by an immobilized mutant of the promiscuous hydrolase/acyltransferase (PestE) (from the thermophilic archaeon Pyrobaculum calidifontis VA1). The HY produced was then acetylated using PestE immobilized on magnetic nanoparticles in a multiphase MBR (conversion: 98%) and simultaneously extracted (extraction: 98%) in ethyl acetate. The work demonstrates that continuous cascade enzymatic reactions can be engineered using artificial membranes to tailor enzyme compartmentalization, mass transport and phase contact according to reaction requirements. Besides, environmental factors proved the sustainability of the integrated membrane bioreactive system.

Freezing and bioreactor in the low-concentration detergents: A novel approach in the decellularization of small-diameter arteries.

Using decellularized small-diameter vascular bypass substitutes (<6 mm) is an efficient method for bypass grafting. A solution containing 0.5% SDS (weight/volume) is commonly used for extended periods to generate acellular tissues. However, this solution causes damage to the microfibril structure and alters the mechanical forces. Hence, the objective of this study is to reduce the concentration of SDS to preserve the structure and achieve efficient decellularization. The study employs a diluted solution of 0.3% SDS (weight/volume) to treat fresh and frozen swine small-diameter arteries, utilizing physical methods such as freezing and thawing. The effectiveness of cell removal was evaluated using histological analysis and the remaining DNA content of the sample. Furthermore, the acellular circuit also assesses the cytotoxicity and proliferation of HUVECs to gauge their safety. Through the use of 0.3% SDS, a bioreactor system, and freezing-thawing, the pig arteries are successfully decellularized, resulting in residual DNA levels of less than 50 ng/mg dry weight. This process does not cause any major changes to the biomechanical or structural properties of the arteries. The acellular samples exhibit no toxicity on the L929 cell line and promote the growth of HUVECs at their highest rate on the fourth day. This allows for the placement of acellular vascular grafts to evaluate physiological processes within the animal body. This is an important requirement in clinical blood vessel transplantation.

Reducing wastewater nitrogen loading by >90% with carbon-amended septic systems: A field demonstration in Barnstable (Cape Cod), Massachusetts.

Onsite wastewater treatment systems (OWTS) are a major source of excess nutrients and co-pollutants in watersheds across the United States. In Barnstable County (Cape Cod), Massachusetts, effluent from septic systems and cesspools contributes approximately 80% of the controllable reactive nitrogen (N) load to numerous impaired estuaries and degrades water quality in the region's sole source aquifer, streams and ponds. In unsewered areas, wastewater N loads could be reduced substantially by Innovative/Alternative (I/A) septic systems designed for enhanced removal. Use, however, has been partly limited by the availability of high performing, cost effective options, while conventional septic systems continue to be installed in watersheds with well documented N impairments. This paper describes the strategic replacement of residential OWTS with two I/A models that incorporate woodchip bioreactors to enhance N removal. Systems were installed at 14 neighboring homes in Barnstable, MA, and monitored for field performance. Influent and effluent were sampled monthly and analyzed for N and phosphorus (P), among other water quality indicators. Flow to each system was continuously metered to estimate nutrient loads. Results from the first 25 months of monitoring for 13 systems with at least a full year of data are presented in terms of 1) reductions in nutrient concentrations and mass loads and 2) reliability of the systems for meeting a performance goal of total N (TN) < 10 mg/L. Discussion supports consideration of where and how these technologies may be successfully used to manage excess N in sensitive watersheds.

Monitoring small-scale bioreactor studies for media development using polarized total synchronous fluorescence spectroscopy (pTSFS) and synchronous light scattering (SyLS).

Biopharmaceutical process development often involves the use of small-scale bioreactors (SSBR) for optimizing media formulations and process conditions during scale up to commercial scale production. Two key process parameters (CPP) used in SSBR studies are protein titre and viable cell density (VCD). Here, we explore the efficacy of parallel polarized total synchronous fluorescence spectroscopy (TSFS||) and Synchronous Light Scattering (SyLS||) to qualitatively monitor these CPPs and quantitatively predict titre and VCD for a large-scale cell culture media optimization SSBR study. The study involved 71 different media formulations (50+ components each), and the bioprocess was run for 13 days or more. Samples were extracted at set times (Day 0, 3, 9, and 13) and clarified by centrifugation. TSFS|| spectra showed significant emission changes along with increased light scatter over the course of the bioprocess. SyLS|| measurements strongly correlated with particle size data obtained from Dynamic Light Scattering but did not correlate well with VCD probably because of the centrifugation-based sample preparation. Statistical and principal component analysis (PCA) of the pTSFS data showed that spectral variation was greater between media formulations than due to the evolving bioprocess. This prevented the development of accurate global prediction models for media performance (e.g., predicting product titre at day 9 from media spectra measured at day 0). However, classification methods were successfully used to select media subsets with better quantitative prediction accuracy based on spectral similarities. A practical binary (high/low performance) classification model based on Support Vector Machines was generated for media formulation screening. Combining emission and scatter measurements with multivariate data analysis provides a more holistic, multi-attribute bioprocess monitoring method that minimizes the need to use different offline analytical methods. This methodology can be used to monitor process trajectories and deviations, and ultimately be used to predict bioprocess CPPs when implemented on production scale processes where there is much less compositional variation in the media. We believe this SSBR-pTSFS/SyLS approach will provide a valuable resource to develop the design/parameter space for in-process monitoring at production scale from early-stage process/media development studies.

Perfusion Bioreactor Conditioning of Small-diameter Plant-based Vascular Grafts.

BACKGROUND: Vascular grafts are mainly composed of synthetic materials, but are prone to thrombosis and intimal hyperplasia at small diameters. Decellularized plant scaffolds have emerged that provide promising alternatives for tissue engineering. We previously developed robust, endothelialized small-diameter vessels from decellularized leatherleaf viburnum. This is the first study to precondition and analyze plant-based vessels under physiological fluid flow and pressure waveforms. Using decellularized leatherleaf viburnum as tissue-engineered grafts for implantation can have profound impacts on healthcare due to their biocompatibility and cost-effective production. METHODS: A novel perfusion bioreactor was designed, capable of accurately controlling fluid flow rate and pressure waveforms for preconditioning of small-diameter vascular grafts. A closed-loop system controlled pressure waveforms, mimicking physiological values of 50-120 mmHg at a frequency of 8.75 Hz for fluid flow reaching 5 mL/min. Plant-based vascular grafts were recellularized with endothelial and vascular smooth muscle cells and cultured for up to 3 weeks in this bioreactor. Cell density, scaffold structure and mechanics, thrombogenicity, and immunogenicity of grafts were evaluated. RESULTS: Bioreactor treatment with fluid flow significantly increased luminal endothelial cell density, while pressure waveforms reduced thrombus formation and maintained viable vascular smooth muscle cells within inner layers of grafts compared to static controls. Suture retention of grafts met transplantation standards and white cell viability was suitable for vascular remodeling. CONCLUSION: Low thrombogenicity of endothelialized leatherleaf viburnum holds great potential for vascular repair. This study provides insight into benefits of conditioning plant-based materials with hemodynamic forces at higher frequencies that have not previously been investigated.

Attached and suspended biomass kinetics in an IFAS-MBR system operated under intermittent aeration: Long-term monitoring under SRT variation.

This study thoroughly investigates a Membrane BioReactor - Integrated Fixed Film Activated Sludge - Intermittent Aeration (MBR-IFAS-IA) pilot plant operated from a biokinetic point of view. Specifically, respirometric techniques were applied on suspended and attached biomass to evaluate kinetic and stoichiometric parameters. The main aim was to investigate how the simultaneous presence of biofilm and activated sludge could affect the kinetic behaviour and the role of the Sludge Retention Time (SRT) variation in the kinetic behaviour of the system. The results highlighted a mutual interaction between suspended biomass and biofilm in the IFAS-MBR configuration. In Period I both the heterotrophic yield and growth rate of suspended biomass were higher compared to that of biofilm, thus highlighting higher affinity with organic matter; in contrast, the biofilm showed high affinity with nitrification, with increased nitrification rates with decreasing SRT and sustaining nitrification in the activated sludge due to "seeding" effect. Therefore, the suggestion is that it is possible to operate IFAS-MBR systems at low SRT without hampering the nitrification ability due to the growth of nitrifiers in the biofilm. Respirometry has been confirmed to be an effective tool for evaluating biomass kinetic and stoichiometric parameters. The results of this study highlighted the effect of IFAS configuration and can help apply mathematical models in the design phase and monitor biomass viability during plant operations.

Innovative high-performance and energy-positive Co-treatment of organic kitchen waste and domestic wastewater using a fluidized bed ceramic membrane bioreactor.

The aim of the study was to efficiently treat organic kitchen waste (FW) and domestic wastewater (DWW) together in an anaerobic fluidized bed bioreactor equipped with a ceramic membrane (AnFCMBR) through a sustainable approach considering energy recovery. The system operated continuously for 519 days at room temperature, and different filtration fluxes (1.7 and 5 L/m2/h), hydraulic retention times (HRTs) (22 h and 7 h), and organic loading rate (OLRs) (0.46, 1.52, 3.42, 6.08 kg/m3.d) were tested. The amount of organic matter in DWW may be insufficient for feasible gas production, but this challenge can be resolved through the addition of food waste. Influent chemical oxygen demand (COD) of 500 ± 143 mg/L gradually increased to 2000 ± 196 mg/L by increasing the portion of FW. The COD removal ranged from 92 to 98% throughout the study, with the membrane and the cake layer contributing 5-8% to the performance. Average supernatant SMP and EPS concentrations increased from 5 ± 1 to 45 ± 5 mg COD/L and from 54 ± 7 to 254 ± 26 mg COD/g VSS, respectively, when the highest amount of FW was added to the synthetic wastewater. This significant increase in SMP and EPS concentrations due to the addition of FW negatively impacted the filtration performance. SRF and CST values also increased with rising OLR, especially with the supplementation of synthetic wastewater with FW. After FW started to be mixed with DWW, the methane production increased approximately 5.5 times. With the use of AnFCMBR for the co-treatment of FW and DWW, it is possible to achieve energy-positive treatment with high-quality effluent that can be reused for various applications, such as irrigation. The methane produced provided 12 times more energy than was needed to operate the bioreactor. This is the first study evaluating the co-treatment of FW and DWW in AnFCMBR under varying operational parameters.