Flow Bioreactor Research Articles

Functionalized Metal-Organic Framework for NADH Regeneration by Hydrogen in a Redox Flow Bioreactor.

The electrochemical regeneration of reduced nicotinamide adenine dinucleotide (NADH) using [Rh(Cp*)(bpy)Cl]+ holds significant promise for the industrial synthesis of chiral chemicals. However, challenges persist due to the high consumption of NADH and the limited efficiency of its cyclic regeneration, which currently hinder widespread application. To address these obstacles, based on in-situ growth of 3D ordered metal-organic framework (NU-1000) on the surface of graphite felt, [Rh(Cp*)(bpy)Cl]+ were immobilized on the Zr6 nodes of NU-1000 by solvent-assisted ligand incorporation (SALI), and applied in a flow bioreactor. Moreover, we employ a gas diffusion electrode (GDE) to oxidize H2, providing a clean proton source for the electrochemical regeneration of NADH. Consequently, highly efficient enzymatic electrocatalytic synthesis of L-lactate was achieved when coupled with L-lactate dehydrogenases (LDH) as a model reaction, and the total turnover number (TTN) reached 19600 and 1750 for [Rh(Cp*)(bpy)Cl]+ and NAD+ after 48 h, corresponding to a high turnover frequency (TOF) of 2350 h-1 and 210 h-1 for [Rh(Cp*)(bpy)Cl]+ and NAD+, respectively. This work provides new insights for the construction of efficient enzymatic electrosynthesis systems in industrial production.

Design and construction of a low-cost compressive loading and perfusion flow bioreactor

This article reports the design and construction of an open-source compressive loading and perfusion flow bioreactor for under $4000, as well as validation of the device and an example use-application. The bioreactor is capable of recording applied force and displacement as well as regulating media flow rate. This bioreactor was built to be user friendly, widely adaptable for modular changes, and made of readily available materials.

Defective flow space limits the scaling up of turbulence bioreactors for platelet generation

To complement donor-dependent platelets supplies, we previously developed an ex vivo manufacturing system using induced pluripotent stem cell (iPSC)-derived expandable immortalized megakaryocyte progenitor cell lines (imMKCLs), and a turbulent flow bioreactor to generate iPSC-derived platelets products (iPSC-PLTs). However, the tank size of the bioreactor was limited to 10 L. Here we examined the feasibility of scaling up to 50 L with reciprocal motion by two impellers. Under optimized turbulence parameters corresponding to 10 L bioreactor, 50 L bioreactor elicited iPSC-PLTs with intact in vivo hemostatic function but with less production efficiency. This insufficiency was caused by increased defective turbulent flow space. A computer simulation proposed that designing 50 L turbulent flow bioreactor with three impellers or a new bioreactor with a modified rotating impeller and unique structure reduces this space. These findings indicate that large-scale iPSC-PLTs manufacturing from cultured imMKCLs requires optimization of the tank structure in addition to optimal turbulent energy and shear stress.

Application of Recycled Filling to Improve the Purification Performance of Confectionery Wastewater in a Vertical Anaerobic Labyrinth Flow Bioreactor

Anaerobic wastewater treatment is, in many cases, a justified alternative to typical activated sludge processes, from a technological, economic, and ecological point of view. The optimisation of fermentation reactors is primarily concerned with increasing the biodegradation of organic compounds and biogas production, as well as improving efficiency in the removal of nitrogen and phosphorus compounds. The aim of the research was to determine the impact of using low-cost recycled filling on the efficiency of treating real confectionery wastewater in a vertical anaerobic labyrinth flow bioreactor. The experiments focused on selecting the organic loading rate that would allow for the effective biodegradation and removal of pollutants, as well as the efficient production of biomethane. It was found that the tested reactor can operate efficiently at a maximum organic loading rate (OLR) of 7.0–8.0 g of chemical oxygen demand (COD)/L·d. In this OLR range, high efficiency was guaranteed for both wastewater treatment and biogas production. However, increasing the OLR value to 8.0 g COD/L·d had a significant negative effect on the methane (CH4) content in the biogas. The most efficient variants achieved a biodegradation efficiency of around 90% of the organic compounds, a CH4 content of over 70% in the biogas, and a biogas yield of over 400 L/kg of COD removed. A significant influence of the applied OLR on the ratio of free organic acids (FOS) to total alkaline capacity (TAC) and pH was observed, as well as a strong correlation of these indicators with the specific biogas yield and CH4 content. The application of a solution based on the use of a hybrid system of anaerobic granulated sludge and an anaerobic filter resulted in an efficient treatment process and an almost complete elimination of suspensions from the wastewater.

Wall-Immobilized Biocatalyst vs. Packed Bed in Miniaturized Continuous Reactors: Performances and Scale-Up.

Sustainable biocatalysis syntheses have gained considerable popularity over the years. However, further optimizations - notably to reduce costs - are required if the methods are to be successfully deployed in a range of areas. As part of this drive, various enzyme immobilization strategies have been studied, alongside process intensification from batch to continuous production. The flow bioreactor portfolio mainly ranges between packed bed reactors and wall-immobilized enzyme miniaturized reactors. Because of their simplicity, packed bed reactors are the most frequently encountered at lab-scale. However, at industrial scale, the growing pressure drop induced by the increase in equipment size hampers their implementation for some applications. Wall-immobilized miniaturized reactors require less pumping power, but a new problem arises due to their reduced enzyme-loading capacity. This review starts with a presentation of the current technology portfolio and a reminder of the metrics to be applied with flow bioreactors. Then, a benchmarking of the most recent relevant works is presented. The scale-up perspectives of the various options are presented in detail, highlighting key features of industrial requirements. One of the main objectives of this review is to clarify the strategies on which future study should center to maximize the performance of wall-immobilized enzyme reactors.

Enhancement of hydrazine accumulation in anammox bioreactors

The role of hydrazine (N2H4) in anammox metabolism has been widely studied; however, studies on N2H4 biosynthesis by anammox bacteria are limited in the literature. In this context, the current research aims to investigate the enhancement of biological N2H4 production in the anammox process in a long-term manner. The experimental studies started with the optimization of the operating conditions to achieve maximum N2H4 accumulation. Under favorable conditions (pH = 8.97 ± 0.08; T = 35.5 ± 0.5 °C; initial hydroxylamine dose = 1.46 ± 0.01 mM), 17.16 ± 0.64 mg L−1 of N2H4 accumulated in the batch systems. The continuity of N2H4 bioproduction was then evaluated by long-term observations. A continuous flow bioreactor was operated in four consecutive manipulated periods under optimized conditions. In the long-term operated bioreactor, 55.10 ± 0.30 mg L−1 N2H4 was accumulated at optimal conditions, which was 2.5 times higher than reported in the literature. Although manipulation of the bioreactor operating conditions initially resulted in a significant increase in N2H4 bioaccumulation, it subsequently caused a severe deterioration in anammox activity. However, this could be mitigated by increasing the biomass concentration in the anammox systems. In addition, the relative abundance of Candidatus Kuenenia decreased by 1.88% throughout the long-term operation.

A comprehensive study of glucose and oxygen gradients in a scaled-down model of recombinant HuGM-CSF production in thermoinduced Escherichia coli fed-batch cultures

The effect of gradients of elevated glucose and low dissolved oxygen in the addition zone of fed-batch E. coli thermoinduced recombinant high cell density cultures can be evaluated through two-compartment scale-down models. Here, glucose was fed in the inlet of a plug flow bioreactor (PFB) connected to a stirred tank bioreactor (STB). E. coli cells diminished growth from 48.2 ± 2.2 g/L in the stage of RP production if compared to control (STB) with STB-PFB experiments, when residence time inside the PFB was 25 s (34.1 ± 3.5 g/L) and 40 s (25.6 ± 5.1 g/L), respectively. The recombinant granulocyte-macrophage colony-stimulating factor (rHuGM-CSF) production decreased from 34 ± 7% of RP in inclusion bodies (IB) in control cultures to 21 ± 8%, and 7 ± 4% during the thermoinduction production phase when increasing residence time inside the PFB to 25 s and 40 s, respectively. This, along with the accumulation of acetic and formic acid (up to 4 g/L), indicates metabolic redirection of central carbon routes through metabolic flow and mixed acid fermentation. Special care must be taken when producing a recombinant protein in heat-induced E. coli, because the yield and productivity of the protein decreases as the size of the bioreactors increases, especially if they are carried at high cell density.

Optimization of Process for Enzymatic Resolution of Racemic Amines using Continuous Flow Bioreactor

The optimization of enzymatic process for the resolution of chemically and pharmaceutically important racemic amines is described using a continuous flow bioreactor. Also described are the isolation and purification methods of the reaction products and the various parameters affecting the optimal reaction conditions for the enzymatic resolution to afford the products in high chemical purity and enantiomeric excess (ee) of over 90%.

Shear Stress Quantification in Tissue Engineering Bioreactor Heart Valves: A Computational Approach.

Tissue-engineered heart valves can grow, repair, and remodel after implantation, presenting a more favorable long-term solution compared to mechanical and porcine valves. Achieving functional engineered valve tissue requires the maturation of human cells seeded onto valve scaffolds under favorable growth conditions in bioreactors. The mechanical stress and strain on developing valve tissue caused by different pressure and flow conditions in bioreactors are currently unknown. The aim of this study is to quantify the wall shear stress (WSS) magnitude in heart valve prostheses under different valve geometries and bioreactor flow rates. To achieve this, this study used fluid-structure interaction simulations to obtain the valve's opening geometries during the systolic phase. These geometries were then used in computational fluid dynamics simulations with refined near-wall mesh elements and ranges of prescribed inlet flow rates. The data obtained included histograms and regression curves that characterized the distribution, peak, and median WSS for various flow rates and valve opening configurations. This study also found that the upper region of the valve near the commissures experienced higher WSS magnitudes than the rest of the valve.

Effects of glucose and lactate on Streptococcus mutans abundance in a novel multispecies oral biofilm model.

The oral microbiome plays an important role in protecting oral health. Here, we established a controlled mixed-species in vitro biofilm model and used it to assess the impact of glucose and lactate on the ability of Streptococcus mutans, an acidogenic and aciduric species, to compete with commensal oral bacteria. A chemically defined medium was developed that supported the growth of S. mutans and four common early colonizers of dental plaque: Streptococcus gordonii, Actinomyces oris, Neisseria subflava, and Veillonella parvula. Biofilms containing the early colonizers were developed in a continuous flow bioreactor, exposed to S. mutans, and incubated for up to 7 days. The abundance of bacteria was estimated by quantitative polymerase chain reaction (qPCR). At high glucose and high lactate, the pH in bulk fluid rapidly decreased to approximately 5.2, and S. mutans outgrew other species in biofilms. In low glucose and high lactate, the pH remained above 5.5, and V. parvula was the most abundant species in biofilms. By contrast, in low glucose and low lactate, the pH remained above 6.0 throughout the experiment, and the microbial community in biofilms was relatively balanced. Fluorescence in situ hybridization confirmed that all species were present in the biofilm and the majority of cells were viable using live/dead staining. These data demonstrate that carbon source concentration is critical for microbial homeostasis in model oral biofilms. Furthermore, we established an experimental system that can support the development of computational models to predict transitions to microbial dysbiosis based on metabolic interactions.IMPORTANCEWe developed a controlled (by removing host factor) dynamic system metabolically representative of early colonization of Streptococcus mutans not measurable in vivo. Hypotheses on factors influencing S. mutans colonization, such as community composition and inoculation sequence and the effect of metabolite concentrations, can be tested and used to predict the effect of interventions such as dietary modifications or the use of toothpaste or mouthwash on S. mutans colonization. The defined in vitro model (species and medium) can be simulated in an in silico model to explore more of the parameter space.

A continuous flow bioreactor system for high-throughput hyperpolarized metabolic flux analysis.

Hyperpolarized carbon-13 labeled compounds are increasingly being used in medical MR imaging (MRI) and MR imaging (MRI) and spectroscopy (MRS) research, due to its ability to monitor tissue and cell metabolism in real-time. Although radiological biomarkers are increasingly being considered as clinical indicators, biopsies are still considered the gold standard for a large variety of indications. Bioreactor systems can play an important role in biopsy examinations because of their ability to provide a physiochemical environment that is conducive for therapeutic response monitoring ex vivo. We demonstrate here a proof-of-concept bioreactor and microcoil receive array setup that allows for ex vivo preservation and metabolic NMR spectroscopy on up to three biopsy samples simultaneously, creating an easy-to-use and robust way to simultaneously run multisample carbon-13 hyperpolarization experiments. Experiments using hyperpolarized [1-13C]pyruvate on ML-1 leukemic cells in the bioreactor setup were performed and the kinetic pyruvate-to-lactate rate constants ( ) extracted. The coefficient of variation of the experimentally found s for five repeated experiments was . With this statistical power, treatment effects of 30%-40% change in lactate production could be easily differentiable with only a few hyperpolarization dissolutions on this setup. Furthermore, longitudinal experiments showed preservation of ML-1 cells in the bioreactor setup for at least 6 h. Rat brain tissue slices were also seen to be preserved within the bioreactor for at least 1 h. This validation serves as the basis for further optimization and upscaling of the setup, which undoubtedly has huge potential in high-throughput studies with various biomarkers and tissue types.

Study of parameters for the design of a continuous flow laboratory biogas reactor

This study aimed to determine the parameters needed to design a continuos flow bioreactor. Developing studies that expand the quantity and efficiency of biogas currently produced on an industrial anaerobic wastewater treatment in a food processing plant in the Brazilian State of Goiás. The procedures developed in this work we used nine sets of bioreactors in batch and followed the German Standard VDI 4630. A special workbench was developed for that purpose, alternative procedures for determining the volume of the produced biogas were tested. The biomass studied in this work is the resulting sludge of the wastewater treatment from an industrial plant. The daily production is about nine tons, currently, which is totally discarded. Through this batch tests, it was possible to calculate the volume of biogas production and the ideal biomass retention time of the biomass. Obtained data to project a laboratory continuous flow bioreactor. With this data was made a projection of the biogas production potential by the use of this feedstock in an industrial bioreactor constructed for this purpose.

Mechanical factors influence β-catenin localization and barrier properties.

Mechanical forces are of major importance in regulating vascular homeostasis by influencing endothelial cell behavior and functions. Adherens junctions are critical sites for mechanotransduction in endothelial cells. β-catenin, a component of adherens junctions and the canonical Wnt signaling pathway, plays a role in mechanoactivation. Evidence suggests that β-catenin is involved in flow sensing and responds to tensional forces, impacting junction dynamics. The mechanoregulation of β-catenin signaling is context-dependent, influenced by the type and duration of mechanical loads. In endothelial cells, β-catenin's nuclear translocation and signaling are influenced by shear stress and strain, affecting endothelial permeability. The study investigates how shear stress, strain, and surface topography impact adherens junction dynamics, regulate β-catenin localization, and influence endothelial barrier properties. Insight box Mechanical loads are potent regulators of endothelial functions through not completely elucidated mechanisms. Surface topography, wall shear stress and cyclic wall deformation contribute overlapping mechanical stimuli to which endothelial monolayer respond to adapt and maintain barrier functions. The use of custom developed flow chamber and bioreactor allows quantifying the response of mature human endothelial to well-defined wall shear stress and gradients of strain. Here, the mechanoregulation of β-catenin by substrate topography, wall shear stress, and cyclic stretch is analyzed and linked to the monolayer control of endothelial permeability.

Bioreactor Design for Culturing Vascularized Engineered Tissue in Flow Conditions.

Background: Current treatments for congenital heart defects often require surgery and implantation of a synthetic patch or baffle that becomes a fibrous scar and leads to a high number of reoperations. Previous studies in rats have shown that a prevascularized scaffold can integrate into the heart and result in regions of vascularized and muscularized tissue. However, increasing the thickness of this scaffold for use in human hearts requires a method to populate the thick scaffold and mature it under physiologic flow and electrical conditions. Experiment: We developed a bioreactor system that can perfuse up to six 7 mm porous scaffolds with tunable gravity-mediated flow and chronic electrical stimulation. Three polymers, which have been reported to be biocompatible, were evaluated for effects on the viability of induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM). Bioreactor flow and electrical stimulation functions were tested, and the bioreactor was operated for up to 7 days to ensure reliability and lack of leaks in a 37°C, humidified incubator. Height and flow relationships were measured for perfusion through an electrospun polycaprolactone and gelatin scaffold, previously reported by our laboratory. Culture with cells was evaluated by plating human umbilical vein endothelial cells and human dermal fibroblasts on top of the scaffolds in both static and flow conditions for 2, 5, and 7 days. As a proof-of concept, scaffolds were cryosectioned and cell infiltration was quantified using immunofluorescence staining. Results: Neither MED610 (Stratasys), Vero (Stratasys), nor FORMLAB materials affected the viability of iPSC-CM, and MED610 was chosen for manufacture due to familiarity of 3D printing from this material. The generation of electrical field stimulation from 0 to 5 V and physiological ranges of pump capacities were verified. The relationship between height and flow was calculated for scaffolds with and without cells. Finally, we demonstrated evaluation of cell depth and structure in scaffolds cultured for 2, 5, and 7 days. Conclusion: The gravity-mediated flow bioreactor system we developed can be used as a platform for 3D cell culture particularly designed for perfusing vascularized tissue constructs with electrical stimulation for cardiac maturation.

Treatment of 2-naphthalene sulfonate by free and immobilized cells of indigenous isolate Comamonas sp. C2 and toxicity evaluation of biotransformed sample

An indigenous bacterial strain Comamonas sp. C2, capable of utilizing 2-naphthalene sulfonate (2-NS) as a sole source of carbon, was isolated after enrichment of microbial diversity present in sample collected from sewage treatment plant (STP) receiving untreated or partially treated effluent from textile industries. The isolate was able to degrade 100 mg/L of 2-NS, when grown in sulfur free minimal salt medium (SFMSM), after an incubation of 8 h. The release of sulfite/sulfate ions during biotransformation, assay of protocatechuate 4, 5-dioxygenase activity and formation of metabolic intermediates like salicylaldehyde, protocatechuate and 2-hydroxy-4-carboxy-muconatesemialdehyde in the biologically treated sample indicated to the meta-cleavage of aromatic ring of 2-NS. An immobilized-cell plug flow bioreactor (PFR) was developed and evaluated for its efficiency to treat simulated feed supplemented with 100 mg/L of 2-NS. The cells of C2 were immobilized on pieces of polyurethane foam (PUF) and refractory brick pieces (RBP). The PFR, when operated at an HRT of 36 h, achieved complete degradation of 100 mg/L of 2-NS corresponding to 89% lowering of COD. The reduction in overall toxicity of 2-NS after biological treatment was evaluated using plant (Triticum aestivum and Vigna radiata) and animal (freshwater fish Channa punctatus) models, exposed to both untreated and PFR treated samples. To the best of our knowledge, there are no similar reports on exploitation of the metabolic potential of Comamonas sp. in 2-NS degradation and validation of the biological transformation by toxicity analysis.

Algae Cultivation as Measure for the Sanitation of Organic Waste—A Case Study Based on the Alga Galdieria sulphuraria Grown on Food Waste Hydrolysate in a Continuous Flow Culture

Due to its growth under harsh acidic conditions, the microalga Galdieria sulphuraria may offer the opportunity to combine sanitation and the utilization of organic waste streams. To further deepen the knowledge of alternative waste treatment strategies that allow for holistic utilization, the control and removal of microbial contaminants via non-sterile heterotrophic G. sulphuraria on food waste hydrolysate were investigated in a continuous flow bioreactor culture. Furthermore, a substrate reservoir and harvested biomass were stored under non-sterile conditions over a period of 12 days. Despite the non-sterile conditions, the microbial load of the biomass could be kept under control. Neither the pathogen Salmonella sp. nor the coliform bacteria Escherichia coli could be found. Only nine counts per g of biomass were found for species belonging to Enterococcus spp., Enterobacteriacae, and moulds. Aerobic spore formers were counted with 2700 counts per g of biomass. Most of the aerobic mesophilic counts were formed by yeasts (1.5 × 106 vs. 1.3 × 106 counts per g biomass). The results revealed that, when using acidic growth conditions, contamination will not take over the culture; thus, the sterilization of waste materials can be skipped. It is assumed that such an approach can result in efficient processes for future waste-based bioeconomy strategies.

Environmental ammonia analysis based on exclusive nitrification by nitrifying biofilm screened from natural bioresource

Biofilm-based biological nitrification is widely used for ammonia removal, while hasn't been explored for ammonia analysis. The stumbling block is the coexist of nitrifying and heterotrophic microbes in real environment resulting in non-specific sensing. Herein, an exclusive ammonia sensing nitrifying biofilm was screened from natural bioresource, and a bioreaction-detection system for the on-line analysis of environmental ammonia based on biological nitrification was reported. The nitrifying microbes were aggregated into a nitrifying biofilm through a result-oriented bioresource enrichment strategy. The predominant nitrifying population and progressive surface reaction in the plug flow bioreactor led to the exclusive and exhaustive ammonia biodegradation for the establishment of a novel analytical method. The on-line ammonia monitoring prototype achieved complete biodegradation for determining ammonium nitrogen within 5 min and showed exceptional reliability in long-term real sample measurements without frequent calibration. This work offers a low-threshold natural screening paradigm for developing sustainable bioresource-based analytical technologies.

Constructing a continuous-flow bioreactor with co-immobilized KmAKR and BmGDH for synthesizing tert-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate

tert-Butyl 6-cyano-(3R,5R)-dihydroxyhexanoate ((3R,5R)-2), an important precursor of atorvastatin, can be synthesized through asymmetric reduction of tert-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate ((5R)-1) by the aldo-keto reductases (AKRs), coupled with BmGDHs for cofactor regeneration. Herein, we employed glutaraldehyde (GLA) and polyethyleneimine (PEI) resin microsphere to co-immobilize KmAKR and BmGDH. The immobilizate activity of KmAKR&BmGDH@amino resin-PEI peaked at 73 U/g, and 80.7% activity remained after 10 cycles of reuse. Continuous flow bioreactor packed with the KmAKR&BmGDH@amino resin-PEI had ∼3.7-fold higher space-time yield (STY, 374.7 g/L/d) than the batch mode. Moreover, excellent operational stability was achieved, remaining over 95% conversion after 90 h consecutive usage. Hence, a self-sustaining closed-loop bienzymatic platform running in the continuous flow mode was developed.

Basic Technological Parameters of the Activation Process for Two Bioreactor Configurations

Abstract Technological parameters of the activation process in wastewater treatment plant activation systems are analyzed for basic bioreactor configurations, for the chemostat and for the plug flow, both with the recirculation of concentrated activated sludge. The first type of bioreactors represents conventional aerated tanks, but also loop bioreactors. The second type of bioreactors includes, for example, oxidation ditches. The paper presents an evaluation of the basic technological parameters of the activation process for the two mentioned configurations of bioreactor systems with recirculation of concentrated activated sludge. In addition, a new relation for sludge age evaluation is proposed for an activation system with a plug flow bioreactor. The article also provides examples of connections between the values of the technological parameters of the activation process for the two analyzed types of bioreactor systems.

Interstitial fluid flow contributes to prostate cancer invasion and migration to bone; study conducted using a novel horizontal flow bioreactor

Prostate cancer bone metastasis is the leading cause of cancer-related mortality in men in the United States, causing severe damage to skeletal tissue. The treatment of advanced-stage prostate cancer is always challenging due to limited drug treatment options, resulting in low survival rates. There is a scarcity of knowledge regarding the mechanisms associated with the effects of biomechanical cues by the interstitial fluid flow on prostate cancer cell growth and migration. We have designed a novel bioreactor system to demonstrate the impact of interstitial fluid flow on the migration of prostate cancer cells to the bone during extravasation. First, we demonstrated that a high flow rate induces apoptosis in PC3 cells via TGF-β1 mediated signaling; thus, physiological flow rate conditions are optimum for cell growth. Next, to understand the role of interstitial fluid flow in prostate cancer migration, we evaluated the migration rate of cells under static and dynamic conditions in the presence or absence of bone. We report that CXCR4 levels were not significantly changed under static and dynamic conditions, indicating that CXCR4 activation in PC3 cells is not influenced by flow conditions but by the bone, where CXCR4 levels were upregulated. The bone-upregulated CXCR4 levels led to increased MMP-9 levels resulting in a high migration rate in the presence of bone. In addition, upregulated levels of α v β 3 integrins under fluid flow conditions contributed to an overall increase in the migration rate of PC3 cells. Overall, this study demonstrates the potential role of interstitial fluid flow in prostate cancer invasion. Understanding the critical role of interstitial fluid flow in promoting prostate cancer cell progression will enhance current therapies for advanced-stage prostate cancer and provide improved treatment options for patients.