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Apolipoprotein A-I (apoA-I), the primary protein component of plasma high-density lipoproteins (HDL), is comprised of two structural regions, an N-terminal amphipathic α-helix bundle domain (residues 1-184) and a hydrophobic C-terminal domain (residues 185-243). When a recombinant fusion protein construct [bacterial pelB leader sequence - human apoA-I (1-243)] was expressed in Escherichia coli shaker flask cultures, apoA-I was recovered in the cell lysate. By contrast, when the C-terminal domain was deleted from the construct, large amounts of the truncated protein, apoA-I (1-184), were recovered in the culture medium. Consequently, following pelB leader sequence cleavage in the E. coli periplasmic space, apoA-I (1-184) was secreted from the bacteria. When the pelB-apoA-I (1-184) fusion construct was expressed in a 5 L bioreactor, substantial foam production (~30 L) occurred. Upon foam collection and collapse into a liquid foamate, SDS-PAGE revealed that apoA-I (1-184) was the sole major protein present. Incubation of apoA-I (1-184) with phospholipid vesicles yielded reconstituted HDL (rHDL) particles that were similar in size and cholesterol efflux capacity to those generated with full-length apoA-I. Mass spectrometry analysis confirmed that pelB leader sequence cleavage occurred and that foam fractionation did not result in unwanted protein modifications. The facile nature and scalability of bioreactor-based apolipoprotein foam fractionation provide a novel means to generate a versatile rHDL scaffold protein.

Compartments are a fundamental feature of life, based variously on lipid membranes, protein shells, or biopolymer phase separation. Here, this combines self-assembling bacterial microcompartment (BMC) shell proteins and liquid-liquid phase separation (LLPS) to develop new forms of compartmentalization. It is found that BMC shell proteins assemble at the liquid-liquid interfaces between either 1) the dextran-rich droplets and PEG-rich continuous phase of a poly(ethyleneglycol)(PEG)/dextran aqueous two-phase system, or 2) the polypeptide-rich coacervate droplets and continuous dilute phase of a polylysine/polyaspartate complex coacervate system. Interfacial protein assemblies in the coacervate system are sensitive to the ratio of cationic to anionic polypeptides, consistent with electrostatically-driven assembly. In both systems, interfacial protein assembly competes with aggregation, with protein concentration and polycation availability impacting coating. These two LLPS systems are then combined to form a three-phase system wherein coacervate droplets are contained within dextran-rich phase droplets. Interfacial localization of BMC hexameric shell proteins is tunable in a three-phase system by changing the polyelectrolyte charge ratio. The tens-of-micron scale BMC shell protein-coated droplets introduced here can accommodate bioactive cargo such as enzymes or RNA and represent a new synthetic cell strategy for organizing biomimetic functionality.

Lower back pain continues to be a global epidemic, limiting quality of life and ability to work, due in large part to symptomatic disc degeneration. Development of more effective and less invasive biological strategies are needed to treat disc degeneration. In vitro models such as macro- or micro-bioreactors or mechanically active organ-chips hold great promise in reducing the need for animal studies that may have limited clinical translatability, due to harsher and more complex mechanical loading environments in human discs than in most animal models. This review highlights the complex loading conditions of the disc in situ, evaluates state-of-the-art designs for applying such complex loads across multiple length scales, from macro-bioreactors that load whole discs to organ-chips that aim to replicate cellular or engineered tissue loading. Emphasis was placed on the rapidly evolving more customizable organ-chips, given their greater potential for studying the progression and treatment of symptomatic disc degeneration. Lastly, this review identifies new trends and challenges for using organ-chips to assess therapeutic strategies.

Textile wastewater poses a significant environmental challenge, primarily due to the presence of diverse contaminants, especially textile dyes. Untreated release of these effluents directly into aquatic systems can lead to esthetic degradation, eutrophication, reduced photosynthetic activity, and accumulation of hazardous substances. Although conventional treatment methods are employed for reducing the contaminant load in effluents, they often are less efficient, thus prompting the exploration of innovative alternatives. Current review highlights myco-remediation as an inexpensive, promising and environmentally sustainable solution. Fungi, with their diverse decontamination mechanisms such as biosorption, biotransformation, and immobilization, prove effective in reducing heavy metals, persistent organic pollutants, and emerging contaminant levels present in these effluents, However, more research effort is needed to apply the biodegradation strategy to decompose completely the "forever chemicals" per‐ and polyfluorinated alkyl substances. Fungi play a key role in degrading and decolorizing textile dyes due to their biocatalytic activity mediated by the production of oxidative enzymes, such as laccases, lignin peroxidases, and manganese peroxidases, as well as their dye adsorption capabilities. This comprehensive review concentrates on fungi-based remediation of textile wastewater effluents, including the mechanisms they employ. While most studies concentrate on effluent treatment, this review also explores the concurrent utilization of biomass and growth kinetics for efficient reduction in pollutant concentrations. Further, the current work showed data on optimization of growth conditions such as pH, temperature and nutrient requirements that lead to efficient effluent decontamination. [ABSTRACT FROM AUTHOR]

This paper proposes an embedded Internet of Things (IoT) system for bioreactor sensor integration, aimed at optimizing temperature and turbidity control during cell cultivation. Utilizing an ESP32 development board, the system makes advances on previous iterations by incorporating superior analog-to-digital conversion capabilities, dual-core processing, and integrated Wi-Fi and Bluetooth connectivity. The key components include a DS18B20 digital temperature sensor, a TS-300B turbidity sensor, and a Peltier module for temperature regulation. Through real-time monitoring and data transmission to cloud platforms, the system facilitates advanced process control and optimization. The experimental results on yeast cultures demonstrate the system's effectiveness at maintaining optimal growth, highlighting its potential to enhance bioprocessing techniques. The proposed solution underscores the practical applications of the IoT in bioreactor environments, offering insights into the improved efficiency and reliability of culture cultivation processes. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. 1UtHR5dBbf2f8WaGLBYUtH Video Abstract [ABSTRACT FROM AUTHOR]

To investigate the cytotoxicity on human dental pulp cells (HDPCs) and Streptococcus mutans (S.mutans) biofilm formation on universal resin composite cements (UCs). Three UCs (RelyX Universal, 3 M Oral Care - RXU; Panavia SA Cement Universal, Kuraray Noritake - PSAU; SoloCem, Coltene - SCM) and one 'gold-standard' multi-step cement (Panavia V5, Kuraray Noritake - PV5) were used following two polymerization protocols (light-cured - LC; self-cured - SC). Cytotoxicity (MTT) tests were performed after 1, 3 and 7 days of direct contact. Carboxy-2′,7′-dichlorodihydrofluorescein diacetate was used to detect the release of reactive oxygen species (ROS), and interleukin 6 (IL-6) expression was analyzed by IL-6 proquantum high sensitivity immunoassay. S. mutans biofilms were grown on UCs samples in a bioreactor for 24 h, then adherent viable biomass was assessed using MTT assay. For microbiological procedures, half of UCs samples underwent accelerated aging. Data were statistically analyzed (α = 0.05). The highest cytotoxicity was observed for PSAU SC, RXU SC, and PV5 SC at day 1, then for SC RXU after 3 days, and SC PSAU, LC PV5 and SCM after 1-week (p < 0.05). There was no increase in IL-6 expression after 1 day, while it increased depending on the group at 3 and 7 days. The highest ROS expression after 12 h was recorded for PSAU SC, PV5 SC and PV5 LC. Biofilm formation was as follows: RXU > > PSAU = PV5 > SCM, while light-curing systematically decreased biofilm formation (≈−33 %). Aging leveled out differences between UCs and between polymerization protocols. The choice of cement brand, rather than category, and polymerization protocol influence cell viability and microbiological behavior. Light-curing is beneficial for reducing the harmful pulpal effect that UCs may possess. [Display omitted] [ABSTRACT FROM AUTHOR]

Ethanol production is a significant industrial bioprocess for energy. The primary objective of this study is to control the process reactor temperature to get the desired product, that is, ethanol. Advanced model‐based control systems face challenges due to model‐process mismatch, but Reinforcement Learning (RL) is a class of machine learning which can help by allowing agents to learn policies directly from the environment. Hence a RL algorithm called twin delayed deep deterministic policy gradient (TD3) is employed. The control of reactor temperature is categorized into two categories namely unconstrained and constrained control approaches. The TD3 with various reward functions are tested on a nonlinear bioreactor model. The results are compared with existing popular RL algorithm, namely, deep deterministic policy gradient (DDPG) algorithm with a performance measure such as mean squared error (MSE). In the unconstrained control of the bioreactor, the TD3 based controller designed with the integral absolute error (IAE) reward yields a lower MSE of 0.22, whereas the DDPG produces an MSE of 0.29. Similarly, in the case of constrained controller, TD3 based controller designed with the IAE reward yields a lower MSE of 0.38, whereas DDPG produces an MSE of 0.48. In addition, the TD3 trained agent successfully rejects the disturbances, namely, input flow rate and inlet temperature in addition to a setpoint change with better performance metrics. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Immense industrial growth contributed to tremendous pollution of the environment from a variety of sources. Bioremediation of different toxicants, such as heavy metals and dyes, is greatly appreciated for its cost-effective and environmentally friendly approach. The bacterial strains isolated from textile dye-contaminated soil samples were used in the study for the removal of the toxic pollutants. The bacterial strains were molecularly identified as E. hormaechei subsp. steigerwaltii and Bacillus sp. B2022 using 16S rRNA sequencing. The preliminary batch study results of the bacterial co-cultures showed a maximum removal efficiency of 88.57 ± 0.005% on mixed dyes and 90.18 ± 0.005% on Cr (VI) respectively. The scale-up laboratory-sized batch reactor showed the results were the maximum removal percentage of 77.68 ± 0.005% with mixed dyes and 52.79 ± 0.005% on Cr (VI) observed for 6 days. The degraded metabolites analyzed by FT-IR revealed the presence of C-H, C = O, C-O, and C–Cl functional groups. The GC–MS study showed the presence of few metabolites such as decane, undecane, 2,4,6-trimethyl decane, butyl tetradecyl ester phthalic acid, and 2,6,10 trimethyl tetradecane. The integrated treatment approach of applying different techniques in a single platform is positively encouraged in the era of urbanization and industrialization. [ABSTRACT FROM AUTHOR]

Copyright of Light Industry Machinery is the property of Light Industry Machinery Editorial Office and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Using seawater for flushing toilets is a potential solution to reduce freshwater consumption in Guam, an island in the Western Pacific Region. However, elevated salinity levels from seawater can affect microbial treatment efficiency in wastewater plants. To study this effect, synthetic wastewater containing 30 mg/L of ammonium-nitrogen was used to evaluate the impact of various salinity levels on ammonium removal efficiency in a continuously operated polyvinyl-alcohol gel bead bioreactor. The bioreactor was incrementally exposed to salinity levels of 0%, 20%, 30%, 50%, 70%, 80%, and 100%, where 0% represented tap water salinity and 100% was equivalent to Guam's seawater salinity. A recovery phase followed the 100% salinity exposure to assess any changes in treatment processes. The total operational period spanned 212 days. Between 0 and 30% salinity, the ammonium-nitrogen removal efficiency averaged 95.0%, indicating no inhibition of nitrification. However, at 50% salinity, the ammonium-nitrogen concentration began to decrease, and a trend that continued up to 80% salinity, resulting in over 60.0% removal efficiency. At 100% salinity, the removal efficiency rapidly dropped from 59.2% to 15.4% within ten days. When the bioreactor returned to 0% salinity, the removal efficiency quickly recovered to 93.7% within five days. This experiment identified 50% salinity as a critical threshold for nitrification efficiency, while 100% salinity was found to be unfavorable for nitrification. [ABSTRACT FROM AUTHOR]

Background: The production of surfactin, an extracellular accumulating lipopeptide produced by various Bacillus species, is a well-known representative of microbial biosurfactant. However, only limited information is available on the correlation between the growth rate of the production strain, such as B. subtilis BMV9, and surfactin production. To understand the correlation between biomass formation over time and surfactin production, the availability of glucose as carbon source was considered as main point. In fed-batch bioreactor processes, the B. subtilis BMV9 was used, a strain well-suited for high cell density fermentation. By adjusting the exponential feeding rates, the growth rate of the surfactin-producing strain, was controlled. Results: Using different growth rates in the range of 0.075 and 0.4 h-1, highest surfactin titres of 36 g/L were reached at 0.25 h-1 with production yields YP/S of 0.21 g/g and YP/X of 0.7 g/g, while growth rates lower than 0.2 h-1 resulted in insufficient and slowed biomass formation as well as surfactin production (YP/S of 0.11 g/g and YP/X of 0.47 g/g for 0.075 h-1). In contrast, feeding rates higher than 0.25 h-1 led to a stimulation of overflow metabolism, resulting in increased acetate formation of up to 3 g/L and an accumulation of glucose due to insufficient conversion, leading to production yields YP/S of 0.15 g/g and YP/X of 0.46 g/g for 0.4 h-1. Conclusions: Overall, the parameter of adjusting exponential feeding rates have an important impact on the B. subtilis productivity in terms of surfactin production in fed-batch bioreactor processes. A growth rate of 0.25 h-1 allowed the highest surfactin production yield, while the total conversion of substrate to biomass remained constant at the different growth rates. [ABSTRACT FROM AUTHOR]

ABSTRACT In this work, the degradation of mono‐ and polyfluorinated phenolic compounds was demonstrated by a series of crude plant peroxidases, including horseradish root (HRP) and six members of the Cucurbita genus. Highly active samples were identified using a library screening approach in which more than 50 crude plant samples were initially evaluated for defluorination activity toward 4‐fluorophenol. The highest concentrations were observed in the HRP, pumpkin skin (PKS), and butternut squash skin (BNS), which consistently gave the highest intrinsic rates of decomposition for all the substrates tested. Although HRP exhibited a significant decrease in activity with increased fluorination of the phenolic substrate, PKS showed only minor reductions. Furthermore, in silico studies indicated that the active site of HRP poorly accommodates the steric bulk of additional fluorines, causing the substrate to dock farther from the catalytic heme and thus slowing the catalysis rate. We propose that the PKS active site might be larger, allowing closer access to the perfluorinated substrate, and therefore maintaining higher activity compared to the HRP enzyme. However, detailed kinetic characterization studies of the peroxidases are recommended. Conclusively, the high catalytic activity of PKS and its high yield per gram of tissue make it an excellent candidate for developing environmentally friendly biocatalytic methods for degrading fluorinated aromatics. Finally, the success of the library approach in identifying highly active samples for polyfluorinated aromatic compound (PFAC) degradation suggests the method may find utility in the quest for other advanced catalysts for PFAS degradation. [ABSTRACT FROM AUTHOR]

Present study aims to enhance protease production by employing locally isolated bacterial strains using animal skin (beef) wastes as a substrate. A proximate analysis of the substrate (skin waste) was carried out to know the percentage of protein, fats, and ash. To understand the particle size, shape, and other structural changes, scanning electron microscope (SEM) and Fourier transform infrared spectroscopy (FTIR) of both untreated and treated samples were performed. The substrate showed 83.81% crude protein, 11.23% crude fat, and 4.96% ash contents. SEM and FTIR analysis revealed changes in treated samples as compared to that untreated skin waste (control). In 140 isolated strains, 60 strains showed clear zone on 1% skim milk agar medium. The selected strains were cultured for protease assay on a production medium. The maximum protease (249.65 IU/ml) producing strain was identified as Bacillus cereus AUST-7 morphologically, biochemically, and based on 16S rDNA gene sequencing. The bioprocess parameters like aeration rate, shaking speed, and incubation period were optimized in lab scale bioreactor of 1.5 L capacity. During scale-up in a bioreactor (1.5 L), 2 volumes of aeration rate per volume of liquid medium per minute (VVM) aeration, shaking speed of 500 rpm, and 24 h of incubation period yielded maximum protease (1917.5 IU/mL) under submerged fermentation. The results depicted that defatted skin waste could be utilized as a potential substrate for the cost-effective production of alkaline protease. [ABSTRACT FROM AUTHOR]

Tissue engineering endeavors to create in vitro constructs that replicate the properties of native tissue, such as skeletal muscle. This study investigated the use of mechanical stimulation to promote myogenic differentiation and enhance the functionality of bioengineered tissues. Specifically, it aimed to facilitate the differentiation of myoblasts within a three-dimensional scaffold using a defined pattern of mechanical stimulation. C2C12 cells were cultured on a collagen-coated PCL microfilament scaffold and subjected to 24 h of uniaxial static strain using a biomechanical stimulation system. Two onset times of stimulation, 72 h and 120 h post-seeding, were evaluated. Cell proliferation, myogenic marker expression, and alterations in cell morphology and orientation were assessed. Results indicate that static strain on the scaffold promoted myoblast differentiation, evidenced by morphological and molecular changes. Notably, strain initiated at 72 h induced an early differentiation stage marked by MyoD expression, whereas stimulation beginning at 120 h led to a mid-stage differentiation characterized by the co-expression of MyoD and Myogenin, culminating in myotube formation. These results highlight the critical influence of myoblast maturity at the time of strain application on the differentiation outcome. This study provides insights that could guide the optimization of mechanical stimulation protocols in tissue engineering applications. [ABSTRACT FROM AUTHOR]

The muscle tendon junction (MTJ) transmits the force generated by the muscle to the tendon and ultimately to the bone. Tears and strains commonly occur at the MTJ where regeneration is limited due poor vascularisation and the complexity of the tissue. Currently treatments for a complete MTJ tear are often unsuccessful. The creation of a tissue engineered MTJ would therefore be beneficial in the development of a novel treatment. In this study, aligned electrospun polycaprolactone fibres were fabricated and human myoblasts and tenocytes were cultured on the scaffold. The effect of 10 % cyclic strain and co-culture of myoblasts and tenocytes on the MTJ formation was investigated. The application of strain significantly increased cell elongation, and MTJ marker gene expression. Co-culture of myoblasts and tenocytes with strain induced higher MTJ marker gene expression compared with myoblasts and tenocytes cultured separately. Paxillin and collagen 22, naturally found in the MTJ, were also produced when cells were combined and grown in a 10 % strain environment. For the first time these results showed that the combination of the strain and co-culture of myoblasts and tenocytes promotes gene expression and production of proteins that are found in the MTJ. [ABSTRACT FROM AUTHOR]

Probiotic bacteria continue to receive increasing attention in the food and feed industries. However, the production of Bifidobacterium and Lactobacillus at an industrial scale is challenging because of specific nutrient requirements and conditions, which are complicated and costly. We developed low-cost culture media by modifying the carbon and nitrogen sources for Bifidobacterium animalis subsp. lactis KMP-H9-01 and Lactobacillus reuteri KMP-P4-S03 from available food grade components. Sucrose (15 g/l) was selected as a suitable carbon source for both strains because it was the most economic and facilitated bacterial growth that was equal to that of glucose. The Bifidobacterium strain required beef extract as a nitrogen source to multiply. The fermentation of both strains using the modified media formula in 5-L and 50-L bioreactors showed that the highest cell counts of L. reuteri and B. animalis subsp. lactis were 9 and 9.8 log CFU/ml after 12–15 h, respectively. The concentration (g/l) ratio between lactate and acetate obtained from B. animalis subsp. lactis was 7:7.4 at 12 h and 11.4:10.6 at 40 h; the ratio was similar at both time points (6.9: 1.1) for L. reuteri. Thus, this economically modified food-grade medium for the large-scale fermentation of two probiotic bacteria was efficient. [ABSTRACT FROM AUTHOR]

Bacteria within the Paenibacillus genus are known to secrete a diverse array of enzymes capable of breaking down plant cell wall polysaccharides. We studied the extracellular xylanolytic activity of Paenibacillus xylanivorans and examined the complete range of secreted proteins when grown on carbohydrate-based carbon sources of increasing complexity, including wheat bran, sugar cane straw, beechwood xylan and sucrose, as control. Our data showed that the relative abundances of secreted proteins varied depending on the carbon source used. Extracellular enzymatic extracts from wheat bran (WB) or sugar cane straw (SCR) cultures had the highest xylanolytic activity, coincidently with the largest representation of carbohydrate active enzymes (CAZymes). Scaling-up to a benchtop bioreactor using WB resulted in a significant enhancement in productivity and in the overall volumetric extracellular xylanase activity, that was further concentrated by freeze-drying. The enzymatic extract was efficient in the deconstruction of xylans from different sources as well as sugar cane straw pretreated by alkali extrusion (SCRe), resulting in xylobiose and xylose, as primary products. The overall yield of xylose released from SCRe was improved by supplementing the enzymatic extract with a recombinant GH43 β-xylosidase (EcXyl43) and a GH62 α-l-arabinofuranosidase (CsAbf62A), two activities that were under-represented. Overall, we showed that the extracellular enzymatic extract from P. xylanivorans, supplemented with specific enzymatic activities, is an effective approach for targeting xylan within lignocellulosic biomass. [ABSTRACT FROM AUTHOR]

In this work, we present investigation on the behavior of bactosomes containing human cytochrome CYP3A4 in comparison with recombinant enzyme. The bactosomes were adsorbed on the surface of screen-printed electrodes modified with didodecyldimethylammonium bromide. We have shown that bactosomes are effective bioreactors in the case of drug metabolic electroenzymatic reactions. The electrocatalytic reaction was conducted at a working potential of − 0.50 ÷ − 0.55 V. Drug conversion was registered by means of measuring of formaldehyde as product of N-demethylation reaction of erythromycin and time-dependent electrochemical oxidation of diclofenac during electrolysis at controlled working potential. The efficiencies of electrocatalysis of erythromycin N-demethylation as well-known cytochrome P450 3A4 substrate in the case of bactosomes containing human cytochrome P450 3A4 was 3.5 ± 0.4 times higher in comparison with highly purified cytochrome P450 3A4-electrode as catalyst. The efficiencies of electrocatalysis of diclofenac 5-hydroxylation in the case of bactosomes were 3.1 ± 0.3 times higher than for purified enzyme. Bactosomes are effective electrochemical enzymatic nanobioreactors with a high electroenzymatic efficiency for metabolite production. [ABSTRACT FROM AUTHOR]

As one of the most important members of the palm family, coconut (Cocos nucifera L.) currently faces a substantial gap between demand and production. Current plantings of this crop are aging, and these traditional varieties are susceptible to several devastating pests and diseases. Consequently, there is an urgent need to replant and expand coconut lands with new, genetically superior varieties. Such replanting cannot be met through the conventional method of seed nut planting, and tissue culture has emerged as a likely solution to address this problem. However, due to certain technical barriers, elevated costs, and a need for improved efficiency, the development of automated and highly efficient tissue culture techniques is yet to be developed. The present research explores the potential of an in vitro temporary immersion system (TIS) to improve the production of somatic embryogenic callus for plantlet regeneration. Results indicated that, in comparison to the conventional agar-based method used to produce coconut somatic embryogenic callus, the TIS method significantly enhanced embryogenic callus production. The optimal biomass of callus for inoculating the TIS was determined to be 0.2 g in each 900 mL vessel and the most favorable embryogenic developmental stage for employing TIS was the globular stage of embryo development. The most effective immersion time to give the highest yield of embryogenic callus was 5 min every 6 h. This foundational research demonstrates that a TIS step is likely to be important to rapidly produce, on a large scale, coconut plantlets to meet the escalating demand for materials for the replanting of coconut lands. [ABSTRACT FROM AUTHOR]

Tannery effluent is a significant contributor to contaminants such as heavy metals within the ecosystem. Effluents generally contain heavy metals, and they also contain more bacteria that can thrive in such an environment. Bioremediation has ancestrally been performed using bacteria; in recent decades, the implementation of "immobilized" bacteria has acquired recognition as an intriguing technique due to manifold assistance. This review systematizes a humongous amount of extant literature on multifarious toxicants that can be tackled with immobilized bacteria. We further explore assorted deterministic facets using immobilized bacteria for environmental remediation with an emphasis on encapsulation in biomaterials and their role in detoxifying toxic compounds. We explore multiple techniques for immobilizing bacteria in numerous complementary arrays incorporating multiple species of bacteria, factors that influence the remediation process, such as bioreactor layouts used in pilot, lab-scale applications. Exploits and drawbacks of using immobilized bacteria in fermenters to treat tannery effluent are also described. The imperishable future aspects, recovery of significant commodities, in addition to bioremediation, represent an important incentive of the immobilized treatment process that makes more cost-effective, legitimate treatment enforcement that is also congruent with the precepts of the bioeconomy. [ABSTRACT FROM AUTHOR]

Copyright of Food & Fermentation Industries is the property of Food & Fermentation Industries and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Production of large amounts of functional NK and CAR-NK cells represents one of the bottlenecks for NK-based immunotherapy. In this study, we developed a largescale, reliable, and practicable NK and CAR-NK production using G-Rex 100M bioreactors, which depend on a gas-permeable membrane technology. This system holds large volumes of medium with enhanced oxygen delivery, creating conditions conducive to large-scale PBNK and CAR-NK expansions for cancer therapy. Both peripheral blood NK cells (PBNKs) and CAR-NKs expanded in these bioreactors retained similar immunophenotypes and exhibited comparable cytotoxicity towards hepatocellular carcinoma (HCC) cells akin to that of NK and CAR-NK cells expanded in G-Rex 6 well bioreactors. Importantly, cryopreservation minimally affected the cytotoxicity of NK cells expanded using the G-Rex 100M bioreactors, establishing a robust platform for scaled-up NK and CAR-NK cell production. This method is promising for the development of “off-the-shelf” NK cells, supporting the future clinical implementation of NK cell immunotherapy. [ABSTRACT FROM AUTHOR]

Red seaweed Gelidium is used for agar extraction, generating large quantities of by-products (red seaweed by-product - GBP). Its carbohydrate and protein-rich composition makes it a suitable substrate to produce value-added compounds through solid-state fermentation (SSF). For the first time, GBP was used in SSF with Aspergillus ibericus and Aspergillus niger for carbohydrases production and protein enrichment. SSF was performed with unsupplemented GBP, supplemented with Mandel salt solution, and mixed at equal dry mass proportion with plant feedstuffs and with the seaweed Ulva rigida. A. niger CECT 2915 was the best strain under SSF obtaining the maximum xylanase activity (498 ± 49Ug-1), in GBP mixed with sunflower cake and the maximum of cellulase activity (382 ± 37Ug-1) in GBP mixed with rapeseed cake. In these mixtures, protein content increased 1.2-fold by SSF, as well as in GBP mixed with rice bran (1.3-fold). Scale-up of SSF using GBP mixed with sunflower cake from flasks to tray-type bioreactors demonstrated SSF reproducibility, achieving similar xylanase and cellulase activities. In the stirred-drum bioreactor, aeration and low agitation favored both enzymes production. Bioprocessing of GBP with plant feedstuffs using SSF was a cost-effective strategy to produce high-value enzymes, valorizing this seaweed by-product. [ABSTRACT FROM AUTHOR]

Summary: The need for therapeutics to treat a plethora of medical conditions and diseases is on the rise and the demand for alternative approaches to mammalian‐based production systems is increasing. Plant‐based strategies provide a safe and effective alternative to produce biological drugs but have yet to enter mainstream manufacturing at a competitive level. Limitations associated with batch consistency and target protein production levels are present; however, strategies to overcome these challenges are underway. In this study, we apply state‐of‐the‐art mass spectrometry‐based proteomics to define proteome remodelling of the plant following agroinfiltration with bacteria grown under shake flask or bioreactor conditions. We observed distinct signatures of bacterial protein production corresponding to the different growth conditions that directly influence the plant defence responses and target protein production on a temporal axis. Our integration of proteomic profiling with small molecule detection and quantification reveals the fluctuation of secondary metabolite production over time to provide new insight into the complexities of dual system modulation in molecular pharming. Our findings suggest that bioreactor bacterial growth may promote evasion of early plant defence responses towards Agrobacterium tumefaciens (updated nomenclature to Rhizobium radiobacter). Furthermore, we uncover and explore specific targets for genetic manipulation to suppress host defences and increase recombinant protein production in molecular pharming. [ABSTRACT FROM AUTHOR]

During adaptive laboratory evolution experiments, any unexpected interruption in data monitoring or control could lead to the loss of valuable experimental data and compromise the integrity of the entire experiment. Most homemade mini-bioreactors are built employing microcontrollers such as Arduino. Although affordable, these platforms lack the robustness of the programmable logic controller (PLC), which enhances the safety and robustness of the control process. Here, we describe the design and validation of a PLC-controlled morbidostat, an innovative automated continuous-culture mini-bioreactor specifically created to study the evolutionary pathways to drug resistance in microorganisms. This morbidostat includes several improvements, both at the hardware and software level, for better online monitoring and a more robust operation. The device was validated employing Escherichia coli, exploring its adaptive evolution in the presence of didecyldimethylammonium chloride (DDAC), a quaternary ammonium compound widely used for its antimicrobial properties. E. coli was subjected to increasing concentrations of DDAC over 3 days. Our results demonstrated a significant increase in DDAC susceptibility, with evolved populations exhibiting substantial changes in their growth after exposure. [ABSTRACT FROM AUTHOR]

A novel, user-friendly bioreactor for the cultivation of cellularised membranes for tissue engineering has been successfully designed, manufactured, and validated. This bioreactor features a culture vessel and a cover, the latter equipped with one or more sidewalls to ensure airtightness in two distinct zones, thereby maintaining sterile conditions. The cover, designed to integrate seamlessly with the culture vessel, includes several ports compatible with commercial connectors. This design allows the introduction of cells and culture medium without requiring the opening of the cover, thus preserving sterility. Additionally, the cover is equipped with flanges that effectively press the membrane against the bottom surface of the culture vessel, preventing it from shrinking or shifting. This ensures that cells can properly adhere to the membrane and proliferate. Manufactured under Good Manufacturing Practice (GMP) conditions, the bioreactor supports cultivation in optimal aseptic environments, thereby preventing external contamination. This feature is critical for the safe transportation of cultivated tissue to clinical settings. Validation tests have confirmed the bioreactor's excellent performance, endorsing its suitability for intended applications in tissue engineering. [ABSTRACT FROM AUTHOR]

Phycocyanobilin (PCB) is a small chromophore found in certain phycobiliproteins, such as phycocyanins (PCs) and allophycocyanins (APCs). PCB, along with other phycobilins (PBs) and intermediates such as biliverdin (BV) or phycoerythrobilin (PEB), is attracting increasing biotechnological interest due to its fluorescent and medicinal properties that allow potential applications in biomedicine and the food industry. This study aims to optimize PCB synthesis in Escherichia coli BL21 (DE3) and scale the process to a pre-industrial level. Parameters such as optimal temperature, inducer concentration, initial OD600, and stirring speed were analyzed in shake flask cultures to maximize PCB production. The best results were obtained at a temperature of 28 °C, an IPTG concentration of 0.1 mM, an initial OD600 of 0.5, and an orbital shaking speed of 260 rpm. Furthermore, the optimized protocol was scaled up into a 2 L bioreactor batch, achieving a maximum PCB concentration of 3.8 mg/L. Analysis of the results revealed that biosynthesis of exogenous PBs in Escherichia coli BL21 (DE3) is highly dependent on the metabolic burden of the host. Several scenarios, such as too rapid growth, high inducer concentration, or mechanical stress, can advance entry into the stationary phase. That progressively halts pigment synthesis, leading, in some cases, to its excretion into the growth media and ultimately triggering rapid degradation by the host. These conclusions provide a promising protocol for scalable PCB production and highlight the main biotechnological challenges to increase the yields of the process. [ABSTRACT FROM AUTHOR]

During physical activities, chondrocytes experience coupled stimulation of hydrostatic pressure (HP) and a transient increase in temperature (T), with the latter varying within a physiological range from 32.5 °C to 38.7 °C. Previous short-term in vitro studies have demonstrated that the combined hydrostatic pressure-thermal (HP-T) stimuli more significantly enhance chondroinduction and chondroprotection of chondrocytes than isolated applications. Interestingly, this combined benefit is associated with a corresponding increase in HSP70 levels when HP and T are combined. The current study therefore explored the indispensable role of HSP70 in mediating the combined effects of HP-T stimuli on chondrocytes. In this mid-long-term study of in vitro engineered cartilage constructs, we assessed chondrocyte responses to HP-T stimuli using customized bioreactor in standard and HSP70-inhibited cultures. Surprisingly, under HSP70-inhibited conditions, the usually beneficial HP-T stimuli, especially its thermal component, exerted detrimental effects on chondrocyte homeostasis, showing a distinct and unfavorable shift in gene and protein expression patterns compared to non-HSP70-inhibited settings. Such effects were corroborated through mechanical testing and confirmed using a secondary cell source. A proteomic-based mechanistic analysis revealed a disruption in the balance between biosynthesis and fundamental cellular structural components in HSP70-inhibited conditions under HP-T stimuli. Our results highlight the critical role of sufficient HSP70 induction in mediating the beneficial effects of coupled HP-T stimulation on chondrocytes. These findings help pave the way for new therapeutic approaches to enhance physiotherapy outcomes and potentially shed light on the elusive mechanisms underlying the onset of cartilage degeneration, a long-standing enigma in orthopedics. [ABSTRACT FROM AUTHOR]

BACKGROUND: Dyes present in textile wastewater can pose various environmental problems, including toxicity, carcinogenicity and mutagenicity, when released into receiving environments. Sulfidogenic bacteria play a crucial role in wastewater treatment by reducing sulfate and producing sulfide through the utilization of organic compounds in water. The resulting sulfide often transfers its electrons to another electron acceptor. This study focused on the treatment of real textile wastewater using an up‐flow sulfidogenic column bioreactor. RESULTS: The reactor was acclimated to sulfate‐reducing conditions when influent chemical oxygen demand and sulfate concentrations were 1742 and 2000 mg L−1, respectively. Subsequently, a gradual transition was made from sulfate‐reducing conditions to dye‐reducing conditions. Throughout the study, the hydraulic retention time was maintained at 1 day. CONCLUSION: The influent dye concentration was 2722 Pt‐Co, and an impressive dye removal efficiency of approximately 90 ± 2% was achieved. This corresponds to a removal rate of 2450 Pt‐Co L−1 day−1. Although the sulfide concentration in the reactor decreased in the last period, investigating the extent to which this sulfide participates in the dye removal may expand the use of sulfidogenic reactors in the treatment of real textile industry wastewater. © 2024 The Author(s). Journal of Chemical Technology and Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry (SCI). [ABSTRACT FROM AUTHOR]

Viola odorata plant extracts are extensively used in indigenous medicine to treat respiratory diseases. V. odorata’s limited availability in nature, due to geographical conditions and adulteration, makes continuous supply of high-quality plant raw material challenging. This results in low-quality plant extracts that can have inconsistent and sometimes adverse effects when used in medicine. As an effective solution, in vitro plant biomass cultivation conserves plant diversity, ensuring high-quality raw materials with uniform quality and quantity. Toward this, the current study focuses on establishing bioprocess strategy to achieve maximum. V. odorata biomass productivity up to bioreactor level. In this context, in vitro culture conditions were rationally optimised, focusing upon both statistical optimization and one-factor-at-a-time (OFAT) approach, for improved biomass productivity. Inoculum density, macronutrient concentration, shaking speed, temperature, pH and light intensity were systematically optimized to achieve the maximum biomass. This led to increased biomass production from 10.2 ± 0.8 g DW L− 1 to 21.7 ± 0.8 g DW L− 1 within 12 days of the batch cultivation period. Optimized culture conditions were implemented in three different types of bioreactors and cultivation in a stirred tank reactor produced ∼ 19.7 g DW L− 1 of maximum biomass within a 12 day batch cultivation period. Further, the metabolite profile of the bioreactor cultivated biomass (alcoholic extract) was compared with that of the natural plant biomass using gas chromatography-mass spectrometry-based metabolomics. The antibacterial activity of the plant extract was tested against three bacteria responsible for respiratory tract infections, and their antioxidant activity was compared with that of Vitamin E, wherein the results demonstrated comparable activity. The novelty of this study is in the fact that the V. odorata plant cells were cultivated in bioreactors for the first time for potential medicinal applications. It provides an alternative method for sustainable production of plant biomass, thereby conserving the natural plant sources.Key message: Bioreactor cultivation of V. odorata plant cells with biomass productivity equivalent to that in shake flask culture, yielding extracts with a similar metabolite profile and associated bioactivity to that in the natural plant extracts. [ABSTRACT FROM AUTHOR]

Abstract Textile wastewater poses a significant environmental challenge, primarily due to the presence of diverse contaminants, especially textile dyes. Untreated release of these effluents directly into aquatic systems can lead to esthetic degradation, eutrophication, reduced photosynthetic activity, and accumulation of hazardous substances. Although conventional treatment methods are employed for reducing the contaminant load in effluents, they often are less efficient, thus prompting the exploration of innovative alternatives. Current review highlights myco-remediation as an inexpensive, promising and environmentally sustainable solution. Fungi, with their diverse decontamination mechanisms such as biosorption, biotransformation, and immobilization, prove effective in reducing heavy metals, persistent organic pollutants, and emerging contaminant levels present in these effluents, However, more research effort is needed to apply the biodegradation strategy to decompose completely the “forever chemicals” per‐ and polyfluorinated alkyl substances. Fungi play a key role in degrading and decolorizing textile dyes due to their biocatalytic activity mediated by the production of oxidative enzymes, such as laccases, lignin peroxidases, and manganese peroxidases, as well as their dye adsorption capabilities. This comprehensive review concentrates on fungi-based remediation of textile wastewater effluents, including the mechanisms they employ. While most studies concentrate on effluent treatment, this review also explores the concurrent utilization of biomass and growth kinetics for efficient reduction in pollutant concentrations. Further, the current work showed data on optimization of growth conditions such as pH, temperature and nutrient requirements that lead to efficient effluent decontamination.

Abstract 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

Abstract Background The production of surfactin, an extracellular accumulating lipopeptide produced by various Bacillus species, is a well-known representative of microbial biosurfactant. However, only limited information is available on the correlation between the growth rate of the production strain, such as B. subtilis BMV9, and surfactin production. To understand the correlation between biomass formation over time and surfactin production, the availability of glucose as carbon source was considered as main point. In fed-batch bioreactor processes, the B. subtilis BMV9 was used, a strain well-suited for high cell density fermentation. By adjusting the exponential feeding rates, the growth rate of the surfactin-producing strain, was controlled. Results Using different growth rates in the range of 0.075 and 0.4 h-1, highest surfactin titres of 36 g/L were reached at 0.25 h-1 with production yields YP/S of 0.21 g/g and YP/X of 0.7 g/g, while growth rates lower than 0.2 h-1 resulted in insufficient and slowed biomass formation as well as surfactin production (YP/S of 0.11 g/g and YP/X of 0.47 g/g for 0.075 h-1). In contrast, feeding rates higher than 0.25 h-1 led to a stimulation of overflow metabolism, resulting in increased acetate formation of up to 3 g/L and an accumulation of glucose due to insufficient conversion, leading to production yields YP/S of 0.15 g/g and YP/X of 0.46 g/g for 0.4 h-1. Conclusions Overall, the parameter of adjusting exponential feeding rates have an important impact on the B. subtilis productivity in terms of surfactin production in fed-batch bioreactor processes. A growth rate of 0.25 h-1 allowed the highest surfactin production yield, while the total conversion of substrate to biomass remained constant at the different growth rates.

In clinical terms, end-stage liver disease is a group of liver diseases that includes advanced liver disease, liver failure, and decompensated cirrhosis. Liver transplantation has been the most effective treatment for cirrhosis. The limited number of available and suitable living liver donors is a significant limitation in liver transplantation. Acute or chronic rejection could be the cause of liver transplant failure. To overcome rejection, usage of the long-term immunosuppressive drug is a standard post-transplant regimen. However, this therapy can increase the risk of severe viral or fungal infection and malignancy. Various attempts were made to address the liver transplant shortage. One of them is liver tissue engineering. This research was conducted with an artificial liver prototype of Stem Cell Hepatic Intuitive Apparatus (SHiNTA) with a perfusion bioreactor, whose manufacturing process is simple in the form of a liver microstructure consisting of differentiated hepatocytes from an hiPSCs from a modification of the Blackford protocol in a liver biologic scaffold. Liver biologic scaffolds were made from pieces of rabbit liver stored in the Stem Cell and Tissue Engineering (SCTE) laboratory, Fakultas Kedokteran Universitas Indonesia, by decellularization. This study aimed to develop the SHiNTA BALs to sustain the viability of hiPSC-derived hepatocytes in the artificial liver prototype. SHiNTA artificial liver prototype with a perfusion bioreactor connected to a perfusion pump with a specific perfusion rate of 2mL/min showed a higher cell count and confluence, with evenly distributed in the extracellular matrix than SHiNTA blood bag with orbital shaker group up to day 7. Furthermore, the SHiNTA artificial liver with perfusion bioreactor showed a positive signal of cell maturation in the scaffold (ASGPR, HNF4- , and CEBP- ) through immunofluorescence.

The muscle tendon junction (MTJ) transmits the force generated by the muscle to the tendon and ultimately to the bone. Tears and strains commonly occur at the MTJ where regeneration is limited due poor vascularisation and the complexity of the tissue. Currently treatments for a complete MTJ tear are often unsuccessful. The creation of a tissue engineered MTJ would therefore be beneficial in the development of a novel treatment. In this study, aligned electrospun polycaprolactone fibres were fabricated and human myoblasts and tenocytes were cultured on the scaffold. The effect of 10 % cyclic strain and co-culture of myoblasts and tenocytes on the MTJ formation was investigated. The application of strain significantly increased cell elongation, and MTJ marker gene expression. Co-culture of myoblasts and tenocytes with strain induced higher MTJ marker gene expression compared with myoblasts and tenocytes cultured separately. Paxillin and collagen 22, naturally found in the MTJ, were also produced when cells were combined and grown in a 10 % strain environment. For the first time these results showed that the combination of the strain and co-culture of myoblasts and tenocytes promotes gene expression and production of proteins that are found in the MTJ.

Plants produce various pigments that not only appear as attractive colors but also provide valuable resources in applications in daily life and scientific research. Biosynthesis pathways for these natural plant pigments are well studied, and most have multiple enzymes that vary among plant species. However, adapting these pathways to animals remains a challenge. Here, we describe successful biosynthesis of betalains, water-soluble pigments found only in a single plant order, Caryophyllales, in transgenic silkworms by coexpressing three betalain synthesis genes, cytochrome P450 enzyme CYP76AD1, DOPA 4,5-dioxygenase, and betanidin 5-O-glucosyltransferase. Betalains can be synthesized in various tissues under the control of the ubiquitous IE1 promoter but accumulate mainly in the hemolymph with yields as high as 274 μg/ml. Additionally, transformed larvae and pupae show a strong red color easily distinguishable from wild-type animals. In experiments in which expression is controlled by the promoter of silk gland-specific gene, fibroin heavy-chain, betalains are found predominantly in the silk glands and can be secreted into cocoons through spinning. Betalains in transformed cocoons are easily recovered from cocoon shells in water with average yields reaching 14.4 μg/mg. These data provide evidence that insects can synthesize natural plant pigments through a complex, multiple enzyme-mediated synthesis pathway. Such pigments also can serve as dominant visible markers in insect transgenesis applications. This study provides an approach to producing valuable plant-derived compounds by using genetically engineered silkworms as a bioreactor.

Induced pluripotent stem cells (iPSCs) have immense potential for use in disease modeling, etiological studies, and drug discovery. However, the current workflow for iPSC generation and maintenance poses challenges particularly during the establishment phase when specialized skills are required. Although three-dimensional culture systems offer scalability for maintaining established iPSCs, the enzymatic dissociation step is complex and time-consuming. In this study, a novel approach was developed to address these challenges by enabling iPSC generation, maintenance, and differentiation without the need for two-dimensional culture or enzymatic dissociation. This streamlined method offers a more convenient workflow, reduces variability and labor for technicians, and opens up avenues for advancements in iPSC research and broader applications.

Tetrandrine, a bioactive active compound mainly found in the roots of Stephania tetrandra, exhibits various pharmacological properties. In vitro hairy root (HR) culture may serve as a promising solution for the extraction of tetrandrine, overcoming the limitations of natural cultivation. The present study describes the consistent production of tetrandrine from S. tetrandra hairy roots induced by different strains of Agrobacterium rhizogenes. Cultivation in woody plant medium (WPM) resulted in the highest HR biomass (0.056 g/petri-dish) and tetrandrine content (7.28 mg/L) as compared to other media. The maximum HR biomass (6.95 g dw/L) and tetrandrine production (68.69 mg/L) were obtained in the fifth week of cultivation. The presence of ammonium nitrate (800 mg/L), calcium nitrate (1156 mg/L), sucrose (20 g/L) and casein (2 g/L) enhanced the tetrandrine production. Moreover, the fed-batch cultivation demonstrated that the NH 4 NO 3 (1200 mg/L) was an important growth limiting factor that yielded the highest tetrandrine amount (119.59 mg/L). The cultivation of hairy roots in a mist trickling bioreactor for eight weeks was less (26.24 mg/L) than in the flask. Despite a lower tetrandrine yield observed in bioreactors compared to flask cultures, refining the growth medium and fine-tuning bioreactor operations hold promise for boosting tetrandrine yield. [Display omitted] • S. tetrandrae hairy roots were established from leaf explant using A. rhizogenes. • Maximum hairy root and tetrandrine production were obtained in WPM medium. • Presence of NH 4 NO 3 (1200 mg/L) yielded highest tetrandrine amount (119.59 mg/L). • Hairy roots growth was attempted in bubble column and mist trickling bioreactors. [ABSTRACT FROM AUTHOR]

Abstract Background Commonly used media for the differentiation of human pluripotent stem cells into cardiomyocytes (hPSC-CMs) contain high concentrations of proteins, in particular albumin, which is prone to quality variations and presents a substantial cost factor, hampering the clinical translation of in vitro-generated cardiomyocytes for heart repair. To overcome these limitations, we have developed chemically defined, entirely protein-free media based on RPMI, supplemented with L-ascorbic acid 2-phosphate (AA-2P) and either the non-ionic surfactant Pluronic F-68 or a specific polyvinyl alcohol (PVA). Methods and Results Both media compositions enable the efficient, directed differentiation of embryonic and induced hPSCs, matching the cell yields and cardiomyocyte purity ranging from 85 to 99% achieved with the widely used protein-based CDM3 medium. The protein-free differentiation approach was readily up-scaled to a 2000 mL process scale in a fully controlled stirred tank bioreactor in suspension culture, producing > 1.3 × 109 cardiomyocytes in a single process run. Transcriptome analysis, flow cytometry, electrophysiology, and contractile force measurements revealed that the mass-produced cardiomyocytes differentiated in protein-free medium exhibit the expected ventricular-like properties equivalent to the well-established characteristics of CDM3-control cells. Conclusions This study promotes the robustness and upscaling of the cardiomyogenic differentiation process, substantially reduces media costs, and provides an important step toward the clinical translation of hPSC-CMs for heart regeneration.

Abstract Background Trichoderma reesei is known for its ability to produce large amounts of extracellular proteins and is one of the most important industrially used filamentous fungus. Xylanase regulator 1 (XYR1) is the master regulator responsible for the activation of cellulase and hemicellulase gene expression under inducing conditions. It has been reported that strains with point mutations in certain areas of xyr1 bypass the need for inducing carbon source, allowing high (hemi)cellulase production even in the presence of glucose. These mutations also change the profile of produced proteins, shifting it more towards xylanase production, and increase the overall protein production in inducing conditions. However, how these mutations alter the metabolism and other cellular processes to cause these changes remains unclear. Results In this study, we aimed to explore changes caused by a point mutation in xyr1 on transcriptomic and metabolic level to better understand the reasons behind the increased protein production in both repressing glucose and inducing lactose conditions. As expected, the expression of many carbohydrate-active enzyme (CAZy) genes was increased in the xyr1 mutant in both conditions. However, their induction was higher under inducing conditions. The xyr1 mutant strain built more biomass and produced more extracellular proteins during growth on lactose compared to the wild type xyr1 strain. Genes involved in oxidoreductive D-galactose catabolism pathway were upregulated in the xyr1 mutant strain, potentially contributing to the more efficient utilization of lactose. In addition to CAZy genes, clustering and enrichment analysis showed over-representation of mitochondria-related Gene Ontology terms in clusters where gene expression was higher in the xyr1 mutant, indicating that mitochondria play a role in the altered metabolic state associated with the xyr1 mutation. Metabolomics revealed that free tyrosine was more abundant in the xyr1 mutant strain in all measured timepoints, whereas multiple fatty acids were less abundant in the mutant strain on glucose. Conclusions The results contribute to more in-depth knowledge on T. reesei physiology growing under inducing and repressing carbon sources and gives new insights on the function of the master regulator XYR1. The vast data generated serve as a source for new targets for improved protein production.

Abstract 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. Conclusion 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.

Due to their immunomodulatory function, mesenchymal stromal cells (MSCs) are a promising therapeutic with the potential to treat neuroinflammation associated with neurodegenerative diseases. This function is mediated by secreted extracellular vesicles (MSC-EVs). Despite established safety, MSC clinical translation has been unsuccessful due to inconsistent clinical outcomes resulting from functional heterogeneity. Current approaches to mitigate functional heterogeneity include ‘priming’ MSCs with inflammatory signals to enhance function. However, comprehensive evaluation of priming and its effects on MSC-EV function has not been performed. Furthermore, clinical translation of MSC-EV therapies requires significant manufacturing scale-up, yet few studies have investigated the effects of priming in bioreactors. As MSC morphology has been shown to predict their immunomodulatory function, we screened MSC morphological response to an array of priming signals and evaluated MSC-EV identity and potency in response to priming in flasks and bioreactors. We identified unique priming conditions corresponding to distinct morphologies. These conditions demonstrated a range of MSC-EV preparation quality and lipidome, allowing us to discover a novel MSC-EV manufacturing condition, as well as gain insight into potential mechanisms of MSC-EV microglia modulation. Our novel screening approach and application of priming to MSC-EV bioreactor manufacturing informs refinement of larger-scale manufacturing and enhancement of MSC-EV function.

Abstract Background There is a significant demand for intermediate-scale bioreactors in academic and industrial institutions to produce cells for various applications in drug screening and/or cell therapy. However, the application of these bioreactors in cultivating hiPSC-derived immune cells and other blood cells is noticeably lacking. To address this gap, we have developed a xeno-free and chemically defined intermediate-scale bioreactor platform, which allows for the generation of standardized human iPSC-derived hematopoietic organoids and subsequent continuous production of macrophages (iPSC-Mac). Methods We describe a novel method for intermediate-scale immune cell manufacturing, specifically the continuous production of functionally and phenotypically relevant macrophages that are harvested on weekly basis for multiple weeks. Results The continuous production of standardized human iPSC-derived macrophages (iPSC-Mac) from 3D hematopoietic organoids also termed hemanoids, is demonstrated. The hemanoids exhibit successive stage-specific embryonic development, recapitulating embryonic hematopoiesis. iPSC-Mac were efficiently and continuously produced from three different iPSC lines and exhibited a consistent and reproducible phenotype, as well as classical functionality and the ability to adapt towards pro- and anti-inflammatory activation stages. Single-cell transcriptomic analysis revealed high macrophage purity. Additionally, we show the ability to use the produced iPSC-Mac as a model for testing immunomodulatory drugs, exemplified by dexamethasone. Conclusions The novel method demonstrates an easy-to-use intermediate-scale bioreactor platform that produces prime macrophages from human iPSCs. These macrophages are functionally active and require no downstream maturation steps, rendering them highly desirable for both therapeutic and non-therapeutic applications.

Abstract Background The gram-positive bacterium Bacillus subtilis is widely used for industrial enzyme production. Its ability to secrete a wide range of enzymes into the extracellular medium especially facilitates downstream processing since cell disruption is avoided. Although various heterologous enzymes have been successfully secreted with B. subtilis, the secretion of cytoplasmic enzymes with high molecular weight is challenging. Only a few studies report on the secretion of cytoplasmic enzymes with a molecular weight > 100 kDa. Results In this study, the cytoplasmic and 120 kDa β-galactosidase of Paenibacillus wynnii (β-gal-Pw) was expressed and secreted with B. subtilis SCK6. Different strategies were focused on to identify the best secretion conditions. Tailormade codon-optimization of the β-gal-Pw gene led to an increase in extracellular β-gal-Pw production. Consequently, the optimized gene was used to test four signal peptides and two promoters in different combinations. Differences in extracellular β-gal-Pw activity between the recombinant B. subtilis strains were observed with the successful secretion being highly dependent on the specific combination of promoter and signal peptide used. Interestingly, signal peptides of both the general secretory- and the twin-arginine translocation pathway mediated secretion. The highest extracellular activity of 55.2 ± 6 µkat/Lculture was reached when secretion was mediated by the PhoD signal peptide and expression was controlled by the PAprE promoter. Production of extracellular β-gal-Pw was further enhanced 1.4-fold in a bioreactor cultivation to 77.5 ± 10 µkat/Lculture with secretion efficiencies of more than 80%. Conclusion For the first time, the β-gal-Pw was efficiently secreted with B. subtilis SCK6, demonstrating the potential of this strain for secretory production of cytoplasmic, high molecular weight enzymes.

Abstract This study introduces an evaluation methodology tailored for bioreactors, with the aim of assessing the stress experienced by algae due to harmful contaminants released from antifouling (AF) paints. We present an online monitoring system equipped with an ultra-sensitive sensor that conducts non-invasive measurements of algal culture's optical density and physiological stage through chlorophyll fluorescence signals. By coupling the ultra-sensitive sensor with flash-induced chlorophyll fluorescence, we examined the dynamic fluorescence changes in the green microalga Chlamydomonas reinhardtii when exposed to biocides. Over a 24-h observation period, increasing concentrations of biocides led to a decrease in photosynthetic activity. Notably, a substantial reduction in the maximum quantum yield of primary photochemistry (FV/FM) was observed within the first hour of exposure. Subsequently, we detected a partial recovery in FV/FM; however, this recovery remained 50% lower than that of the controls. Integrating the advanced submersible sensor with fluorescence decay kinetics offered a comprehensive perspective on the dynamic alterations in algal cells under the exposure to biocides released from antifouling coatings. The analysis of fluorescence relaxation kinetics revealed a significant shortening of the fast and middle phases, along with an increase in the duration of the slow phase, for the coating with the highest levels of biocides. Combining automated culturing and measuring methods, this approach has demonstrated its effectiveness as an ultrasensitive and non-invasive tool for monitoring the physiology of photosynthetic cultures. This is particularly valuable in the context of studying microalgae and their early responses to various environmental conditions, as well as the potential to develop an AF system with minimal harm to the environment.

Neural organoids consist of three-dimensional tissue derived from pluripotent stem cells that could recapitulate key features of the human brain. During the past decade, organoid technology has evolved in the field of human brain science by increasing the quality and applicability of its products. Among them, a novel approach involving the design of neural organoids engineered by mechanical forces has emerged. This review describes previous approaches for the generation of neural organoids, the engineering of neural organoids by mechanical forces, and future challenges for the application of mechanical forces in the design of neural organoids.