Name: Protocollo aggiornato per lassemblaggio e luso dellarray di minibioreattori (MBRA) (Video) | JoVE Journal
Uploaded: 2025-09-05T14:10:00.000+00:00
Duration: 9 min 38 s
Description: Scopri l'assemblaggio e l'uso dell'array di minibioreattori (MBRA) per studiare la dinamica delle comunità microbiche intestinali ad alto rendimento. Questo metodo consente di analizzare i meccanismi di resistenza alla colonizzazione dei patogeni.

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This work was supported by the NIH T32 Molecular Basis of Infectious Disease Predoctoral Fellowship, NIH T32DK007664, and NIH U19AI157981 Microbiome Discovery and Mechanisms to Combat Antibiotic Resistance at Mucosal Surfaces.
The authors thank Hayden Curnyn for his contributions to the design and fabrication of the bioreactor holders and tubing holders used in this system.
Figure 2 and Figure 3 were partially created in&#160;https://BioRender.com

<ol><li>Gilbert, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Current+understanding+of+the+human+microbiome%5BTitle%5D)+AND+%22Nat+Med%22%5BJournal%5D)">Current understanding of the human microbiome.</a> Nat Med. 24 (4), 392-400 (2018).</li>
<li>Sarkar, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Perspective%3A+simple+state+communities+to+study+microbial+interactions%3A+examples+and+future+directions%5BTitle%5D)+AND+%22Front+Microbiol%22%5BJournal%5D)">Perspective: simple state communities to study microbial interactions: examples and future directions.</a> Front Microbiol. 13, 801864(2022).</li>
<li>Van de Wiele, T., Van den Abbeele, P., Ossieur, W., Possemiers, S., Marzorati, M. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aVan+de+Wiele%2C+T+%22The+impact+of+food+bioactives+on+health%3A+in+vitro+and+ex+vivo+models%22">The impact of food bioactives on health: in vitro and ex vivo models</a>. , Springer. Cham. (2015).</li>
<li>McDonald, J. A. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evaluation+of+microbial+community+reproducibility%2C+stability+and+composition+in+a+human+distal+gut+chemostat+model%5BTitle%5D)+AND+%22J+Microbiol+Methods%22%5BJournal%5D)">Evaluation of microbial community reproducibility, stability and composition in a human distal gut chemostat model.</a> J Microbiol Methods. 95 (2), 167-174 (2013).</li>
<li>Feria-Gervasio, D., Denis, S., Alric, M., Brugère, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vitro+maintenance+of+a+human+proximal+colon+microbiota+using+the+continuous+fermentation+system+P-ECSIM%5BTitle%5D)+AND+%22Appl+Microbiol+Biotechnol%22%5BJournal%5D)">In vitro maintenance of a human proximal colon microbiota using the continuous fermentation system P-ECSIM.</a> Appl Microbiol Biotechnol. 91 (5), 1425-1433 (2011).</li>
<li>Auchtung, J. M., Robinson, C. D., Farrell, K., Britton, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((MiniBioReactor+arrays+%28MBRAs%29+as+a+tool+for+studying+C.+difficile+physiology+in+the+presence+of+a+complex+community%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">MiniBioReactor arrays (MBRAs) as a tool for studying C. difficile physiology in the presence of a complex community.</a> Methods Mol Biol. 1476, 235-258 (2016).</li>
<li>Robinson, C. D., Auchtung, J. M., Collins, J., Britton, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Epidemic+Clostridium+difficile+strains+demonstrate+increased+competitive+fitness+compared+to+nonepidemic+isolates%5BTitle%5D)+AND+%22Infect+Immun%22%5BJournal%5D)">Epidemic Clostridium difficile strains demonstrate increased competitive fitness compared to nonepidemic isolates.</a> Infect Immun. 82 (7), 2815-2825 (2014).</li>
<li>Auchtung, J. M., Robinson, C. D., Britton, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cultivation+of+stable%2C+reproducible+microbial+communities+from+different+fecal+donors+using+minibioreactor+arrays+%28MBRAs%29%5BTitle%5D)+AND+%22Microbiome%22%5BJournal%5D)">Cultivation of stable, reproducible microbial communities from different fecal donors using minibioreactor arrays (MBRAs).</a> Microbiome. 3 (1), 42(2015).</li>
<li>Auchtung, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identification+of+simplified+microbial+communities+that+inhibit+Clostridioides+difficile+infection+through+dilution%2Fextinction%5BTitle%5D)+AND+%22mSphere%22%5BJournal%5D)">Identification of simplified microbial communities that inhibit Clostridioides difficile infection through dilution/extinction.</a> mSphere. 5 (4), e00387-e00320 (2020).</li>
<li>Hobson, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((MiniBioReactor+array+%28MBRA%29+in+vitro+gut+model%3A+A+reliable+system+to+study+microbiota-dependent+response+to+antibiotic+treatment%5BTitle%5D)+AND+%22JAC+Antimicrob+Resist%22%5BJournal%5D)">MiniBioReactor array (MBRA) in vitro gut model: A reliable system to study microbiota-dependent response to antibiotic treatment.</a> JAC Antimicrob Resist. 4 (4), dlac077(2022).</li>
<li>Auchtung, T. A., Lerma, A. I., Schuchart, K., Auchtung, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evaluating+effects+of+antibiotics+across+preclinical+models+of+the+human+gastrointestinal+microbiota%5BTitle%5D)+AND+%22Microbiologyopen%22%5BJournal%5D)">Evaluating effects of antibiotics across preclinical models of the human gastrointestinal microbiota.</a> Microbiologyopen. 14 (4), e70030(2025).</li>
<li>Naimi, S., Viennois, E., Gewirtz, A. T., Chassaing, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Direct+impact+of+commonly+used+dietary+emulsifiers+on+human+gut+microbiota%5BTitle%5D)+AND+%22Microbiome%22%5BJournal%5D)">Direct impact of commonly used dietary emulsifiers on human gut microbiota.</a> Microbiome. 9 (1), 66(2021).</li>
<li>Bolyen, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((interactive%2C+scalable+and+extensible+microbiome+data+science+using+QIIME+2%5BTitle%5D)+AND+%22Nat+Biotechnol%22%5BJournal%5D)">interactive, scalable and extensible microbiome data science using QIIME 2.</a> Nat Biotechnol. 37 (8), 852-857 (2019).</li>
<li>Hobson, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+microbiota-dependent+response+to+anticancer+treatment+in+an+in+vitro+human+microbiota+model%3A+a+pilot+study+with+hydroxycarbamide+and+daunorubicin%5BTitle%5D)+AND+%22Front+Cell+Infect+Microbiol%22%5BJournal%5D)">A microbiota-dependent response to anticancer treatment in an in vitro human microbiota model: a pilot study with hydroxycarbamide and daunorubicin.</a> Front Cell Infect Microbiol. 12, 886447(2022).</li>
<li>Jensen, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dietary+fiber+monosaccharide+content+alters+gut+microbiome+composition+and+fermentation%5BTitle%5D)+AND+%22Appl+Environ+Microbiol%22%5BJournal%5D)">Dietary fiber monosaccharide content alters gut microbiome composition and fermentation.</a> Appl Environ Microbiol. 90 (8), e00964-e01024 (2024).</li>
<li>Ghimire, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Positive+synergistic+effects+of+quercetin+and+rice+bran+on+human+gut+microbiota+reduces+Enterobacteriaceae+family+abundance+and+elevates+propionate+in+a+bioreactor+model%5BTitle%5D)+AND+%22Front+Microbiol%22%5BJournal%5D)">Positive synergistic effects of quercetin and rice bran on human gut microbiota reduces Enterobacteriaceae family abundance and elevates propionate in a bioreactor model.</a> Front Microbiol. 12, 751225(2021).</li>
<li>Hu, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Minibioreactor+arrays+to+model+microbiome+response+to+alcohol+and+tryptophan+in+the+context+of+alcohol-associated+liver+disease%5BTitle%5D)+AND+%22NPJ+Biofilms+Microbiomes%22%5BJournal%5D)">Minibioreactor arrays to model microbiome response to alcohol and tryptophan in the context of alcohol-associated liver disease.</a> NPJ Biofilms Microbiomes. 10 (1), 132(2024).</li>
<li>Jens, J. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((MBRA-2%3A+A+modified+chemostat+system+to+culture+biofilms%5BTitle%5D)+AND+%22Microbiol+Spectr%22%5BJournal%5D)">MBRA-2: A modified chemostat system to culture biofilms.</a> Microbiol Spectr. 11 (1), e02928-e03022 (2023).</li>
</ol>

The authors declare no conflicts of interest

This protocol describes the complete assembly and basic operation of a Minibioreactor Array (MBRA) for the high-throughput cultivation of bacterial communities, incorporating several key refinements to the previously published method. The MBRA system remains a versatile and cost-effective tool that allows researchers to cultivate complex microbial ecosystems while supporting numerous experimental replicates in parallel. In this updated version, we introduce improvements that enhance reproducibility, streamline workflow, and reduce contamination risk. These include chemically etched PTFE straws (Figure 2) to prevent detachment, a feed straw on the media line (Figure 2) to minimize biofilm formation, standardized tubing lengths with an accompanying 3D-printed tubing holder (Supplementary File 3) for a more compact and organized setup, and an optimized reuse protocol that eliminates the need for full disassembly between experiments. Together, these refinements represent iterative improvements developed through extensive use of the MBRA system across diverse experimental applications in our laboratory. By addressing both critical assembly steps and practical enhancements, this discussion underscores the utility of the MBRA&#160;as a continuously evolving model system for microbiome research.
The success of the MBRA system relies heavily on the precise assembly and sterilization of components to ensure contamination-free operation. Key steps include the proper fitting of Q-series caps, tubing, and connectors, which facilitate modular assembly and enable media input and waste collection. Ensuring a tight seal between media bottles, waste reservoirs, and bioreactor chambers is essential for preventing leaks and maintaining sterile conditions. Another critical step is the verification of peristaltic pump flow rates prior to experimentation, as inconsistencies can lead to uneven media delivery and may affect microbial growth dynamics. Most multichannel peristaltic pumps that utilize cassettes include an occlusion adjustment mechanism, which should be used to fine-tune the flow rate of each channel. Even with proper calibration, the E-lab tubing remains a primary source of variability. To mitigate this, it is important to visually monitor the frequency and size of media droplets entering each bioreactor chamber during both the initial fill and during the start of experiments. These visual checks allow for early detection of flow rate inconsistencies that may otherwise compromise experimental reproducibility. Table 2 provides troubleshooting strategies for common issues encountered during the assembly and use of MBRAs. These troubleshooting steps ensure reproducibility across experiments and prevent disruptions during long-term cultivation.
Despite its strengths, the MBRA system has certain limitations that must be considered when designing experiments. Unlike more advanced systems, the MBRA lacks active monitoring capabilities, such as real-time optical density (OD) measurements, pH control, and temperature regulation. This absence of active measurement restricts the system&#39;s ability to monitor dynamic changes in microbial growth and metabolic activity in real-time. Furthermore, while the system supports anaerobic cultivation within chambers, it does not include integrated gas control, which may limit applications requiring precise microaerophilic or CO2-enriched environments. For studies requiring such control, alternative systems with built-in gas regulation may be more suitable.
The MBRA system offers key advantages over existing bioreactor models, including high throughput, scalability, and cost-effectiveness, while retaining the ability to cultivate complex bacterial communities under continuous flow to mimic dynamic environments like the human gastrointestinal tract6,8,10. Its compact, modular design allows for simultaneous operation of multiple bioreactors, making it ideal for high-throughput studies such as screening fecal-derived communities for resistance to pathogen invasion9. This modular design provides extensive experimental flexibility: each strip can be supplied by a single media bottle, as demonstrated in this protocol, or by up to six distinct media sources, one for each bioreactor chamber. Working volume is governed by the length of a slim PTFE waste straw inserted into the waste port of each chamber, which sets the liquid height; in this protocol, 25 mm straws maintain a 15&#8239;mL working volume, but volumes between 1-20&#8239;mL can be achieved by trimming or extending the straw. Additionally, shorter feed straws are inserted into the media inlet to direct inflow toward the chamber base, preventing media from dripping down the chamber walls and reducing biofilm formation above the fill line. Pump speeds or pump tubing diameter can also be adjusted to alter the system&#39;s turnover rate. To date, the MBRA system has been widely used to study the functional and compositional changes of microbial communities in response to a variety of factors, including antibiotics10, cancer medication14, and various dietary compounds12,15,16,17 . The simple, modular design makes it ideal for adaptation to various experimental needs. For example, the MBRA has been modified to study biofilms under chemostat-like conditions18, demonstrating its versatility for microbial ecology studies beyond planktonic cultures.
Future iterations of the MBRA system could benefit from additional engineering upgrades that expand its functionality, precision, and throughput potential. One such enhancement is the incorporation of additional ports into each bioreactor chamber. These ports could be used to support active monitoring of environmental parameters such as pH, temperature, gas, or optical density. This would address one of the model&#39;s most significant limitations by allowing real-time feedback and monitoring. Improvements to the chamber or port geometry could facilitate more thorough and accessible cleaning, reducing residue buildup and discoloration and improving long-term reusability. Integration of additional peristaltic pumps with programmable timers would allow for pulsed or diurnal media inputs, better simulating host-associated environments such as feeding cycles in the human gut. Finally, 3D printing with alternative materials, such as chemically resistant, autoclavable polymers, may allow for greater durability and compatibility with a wider range of reagents. Together, these improvements could significantly expand the experimental scope and fidelity of the MBRA platform.
In conclusion, the MBRA provides a powerful, high-throughput platform for cultivating and studying microbial communities under controlled conditions. While it has limitations in active monitoring and pH control, its flexibility, scalability, and cost-efficiency make it an invaluable tool for a wide range of microbiological studies, particularly those requiring high replicability and experimental throughput. Importantly, the system&#39;s modular design and fabrication approach make it inherently adaptable; researchers have and can continue to tailor the MBRA to suit a wide array of experimental objectives. This adaptability ensures that the MBRA can continue to evolve alongside emerging scientific questions and technologies, maintaining its relevance as a versatile platform for microbiome research.

The human microbiome is a complex ecosystem of microorganisms that plays a critical role in numerous physiological processes and profoundly impacts human health. The human microbiome spans many anatomical sites throughout our body, each containing dynamic microbial communities actively interacting in ways we are yet to fully understand1. Expanding our knowledge on the involvement of microbial communities in health and disease is dependent on our understanding of the microbial interactions happening within these environments1. To effectively study and manipulate these intricate systems for therapeutic purposes, a reductionist approach is necessary. Exploring individual microbial interactions using simplified model systems facilitates a better understanding of the full complexity of the microbiome2.
A wide variety of model systems are available to grow human-derived microbial communities. These systems have advanced our understanding of human-associated microbial communities and range from single-stage batch culture to more complex multi-stage continuous-flow systems. Model systems, such as the Simulator of Human Intestinal Microbial Ecosystem3, the Twin-Vessel Single-Stage Chemostat&#160;system4 and the Environment Control System for Intestinal Microbiota5 replicate the physiological conditions of specific anatomical sites and provide close in vitro approximations of microbial environments. However, their adoption by microbiologists is limited because they are costly, require high-level technical expertise to run and maintain, and have limited throughput.
To address these challenges, we developed the Minibioreactor Array (MBRA) system-a continuous-flow, single-stage culture system designed to facilitate the stable growth of microbial communities from diverse sources in a controlled environment6,7,8. The MBRA system stands out from other gut models with its simplicity of assembly and operation, combined with high-throughput capabilities that allow for the simultaneous cultivation of multiple microbial communities, boosting experimental efficiency. Furthermore, the simple and compact nature of this system allows for its operation within anaerobic and microoxic chambers to facilitate the growth of bacteria from anaerobic and hypoxic sites, such as the gastrointestinal and vaginal tract. The versatile nature of this system has been leveraged for the screening of&#160;Clostridioides difficile-resistant gastrointestinal communities9, as well as testing the effects of antibiotics10,11 and dietary substrates12 on microbial communities.
MBRAs are fabricated by 3D printing or additive manufacturing, prioritizing clarity and water resistance in our material choice (see Table of Materials for polymer information). Each array contains six individual chambers, all equipped with ports for media import, waste export, and sample collection. Fresh media is continuously supplied into the system while waste is concurrently extracted, with flow rates precisely controlled by two peristaltic pumps. The contents in the system are constantly agitated using stir bars and a 60-spot stir plate to facilitate homogeneous cultures. The protocol described here is optimized for a 15&#8239;mL working volume per chamber, though each bioreactor can accommodate a range of 1-20&#8239;mL depending on experimental requirements. The peristaltic pump and pump tubing can accommodate flow rates ranging from 0.016 to 2.9&#8239;mL/min, corresponding to turnover rates of approximately 15.63 to 0.09 h, respectively. While the system is compatible with a wide range of media formulations and dietary or nutritional additions, some practical considerations must be accounted for: highly viscous media may require recalibration of flow rates, and the presence of undissolved particles or insoluble components can clog the pump tubing or narrow connectors, particularly at lower flow rates. The modularity of the system allows for rapid and easy tailoring of experiments by adjusting media choice, sample collection, flow rates, and working volume. In conjunction with four 24-channel peristaltic pumps and two 60-spot stir plates, the system can run 48 separate chambers per experiment in a single anaerobic chamber, supporting high-throughput anaerobic screening.
This protocol serves as a visual guide and updated version of a previously published MBRA assembly and operation method developed by our laboratory4. Several key improvements have been incorporated to enhance reproducibility, streamline workflow, and minimize contamination. First, the PTFE straws are now chemically etched to prevent them from detaching and falling into the bioreactor chambers. Second, a media straw has been added to the feed lines to direct media flow into the bottom of the chambers, preventing media from dripping down the chamber walls. This was a known source of biofilm formation. Third, C-flex tubing lengths have been standardized and shortened, and a 3D printed tube holder has been designed to create a more compact, organized setup. Finally, bioreactors are no longer fully disassembled between each use, significantly reducing the time and material costs associated with repeated experiments. These and other incremental refinements reflect iterative optimization based on extensive use of the system across multiple projects in our laboratory.

<table><tbody><tr><td>&amp;nbsp;V-Tap Guide, Standard Sizes 0-80 to 5/8&quot;</td><td>Big Gator Tools</td><td>STD500NP&amp;nbsp;</td><td></td></tr><tr><td>0.22 &amp;mu;m syringe filter</td><td>Fisher</td><td>SLGVR33RS</td><td></td></tr><tr><td>2mag MIXdrive 60 Stirring Drive (drive only)</td><td>2MAG</td><td>MF 41060</td><td></td></tr><tr><td>Air-tite Hypodermic Needles, 22G x3&quot;</td><td>VWR</td><td>89219-274</td><td></td></tr><tr><td>BD 1mL Slip Tip Sterile Syringes Sterile</td><td>Fisher</td><td>14-823-434</td><td></td></tr><tr><td>Bioreactor</td><td>Proto-Labs</td><td>NA</td><td>3D printed out of DMS Somos Watershed Plastic. See supplementary file 1 for template</td></tr><tr><td>Bioreactor Holders</td><td>Proto-Labs</td><td>NA</td><td>3D printed out of PA 12 Black. See supplementary file 2 for template.</td></tr><tr><td>Diba Omnifit Q-Series Solvent Bottle Cap, GL38/38-430 (glass), 2 UNF(F) Ports w/o Valves, Blue</td><td>Cole Parmer</td><td>EW-21942-86</td><td></td></tr><tr><td>Diba Omnifit Tubing, PTFE, 1/8&quot; (3.2 mm) OD x 1.5 mm ID</td><td>Cole Parmer</td><td>EW-21942-76</td><td></td></tr><tr><td>Dibafit Adapter, 1/4&quot;-28 UNF(M) flat bottom to 3.2 mm ID, PEEK</td><td>Cole Parmer</td><td>EW-21941-49</td><td></td></tr><tr><td>IRWIN 12001ZR Tap Wrench #0-1/4&quot; T-Handle</td><td>Amazon</td><td>B00004YOB0</td><td></td></tr><tr><td>Irwin Hanson High Carbon Steel SAE Fraction Tap 1/4 in. 1 pc</td><td>Zoro</td><td>G7695682</td><td></td></tr><tr><td>Loctite Heavy Duty Epoxy Quick Set 8-Fluid Ounce Bottle</td><td>Amazon</td><td>B0044F59N0</td><td></td></tr><tr><td>Male to Male Luer Lock Connector</td><td>Darwin Microfluidics</td><td>DM-MM-LUER-PP&amp;nbsp;</td><td>Alternative: Strategic Applications Inc Male to Male Luer Connector - 10/pk - Fisher - NC9876577</td></tr><tr><td>Masterflex Adapter Fittings, Luer to Luer, Nylon, Avantor</td><td>VWR</td><td>MFLX45502-56</td><td></td></tr><tr><td>Masterflex Fitting, Nylon, Straight, Female Luer to Hose Barb Adapter, 1/16&quot; ID</td><td>VWR</td><td>MFLX45502-00</td><td></td></tr><tr><td>Masterflex Fitting, Nylon, Straight, Female Luer to Hose Barb Adapter, 3/32&quot; ID</td><td>VWR</td><td>MFLX45502-02</td><td></td></tr><tr><td>Masterflex Fitting, Nylon, Straight, Female Luer to Hose Barb Adapters, 1/8&quot;</td><td>VWR</td><td>MFLX45502-04</td><td></td></tr><tr><td>Masterflex Fitting, Nylon, Straight, Male Luer Lock to Hose Barb Adapter, 1/8</td><td>VWR</td><td>MFLX45505-04</td><td></td></tr><tr><td>Masterflex Fitting, Nylon, Straight, Male Luer x 1/4-28 UNF</td><td>VWR</td><td>MFLX45505-82</td><td></td></tr><tr><td>Masterflex Fitting, Polypropylene, Elbow, Female Luer to Female Luer Adapter</td><td>VWR</td><td>MFLX45508-26</td><td></td></tr><tr><td>Masterflex Ismatec Pump Tubing, 2-Stop, Tygon S3 E-Lab, 0.89 mm ID</td><td>VWR</td><td>MFLX96460-26</td><td></td></tr><tr><td>Masterflex Ismatec Pump Tubing, 2-Stop, Tygon S3 E-Lab, 1.14 mm ID</td><td>VWR</td><td>MFLX96460-30</td><td></td></tr><tr><td>Masterflex&amp;reg; Transfer Tubing, C-Flex, Opaque White, 1/8&quot; ID x 1/4&quot; OD; 25 Ft</td><td>VWR</td><td>MFLX06424-67</td><td></td></tr><tr><td>Mohr&amp;rsquo;s Pinchcock for Tubing&amp;nbsp;</td><td>VWR</td><td>470201-374</td><td></td></tr><tr><td>Neoprene Rubber Fender Washers &amp;frac12;&amp;rdquo; OD x &amp;frac14;&amp;rdquo; ID x 1/16&amp;rdquo; Thickness&amp;nbsp;</td><td>Amazon</td><td>B01A29F1R0</td><td></td></tr><tr><td>Precision Seal Rubber Septa</td><td>Sigma Aldrich</td><td>Z553905</td><td></td></tr><tr><td>Spinbar Micro Stir Bars</td><td>VWR</td><td>58948-375</td><td></td></tr><tr><td>Tetra-Etch</td><td>R.S. Hughest Company</td><td>TE-500</td><td></td></tr><tr><td>Tubing Holder</td><td>Proto-Labs</td><td>NA</td><td>3D printed out of PA 12 Black. See supplementary file 3 for template.</td></tr><tr><td>Watson-Marlow 205S Multichannel Cartridge Pump</td><td>Watson-Marlow</td><td>020.3724.00A</td><td>Discountined, alternative: Ismatec IPC Digital Peristaltic Pumps MFLX7800142 - FISHER - 113-200-014 or Masterflex Ismatec IPC Peristaltic Pump, 0.1 to 11.25 rpm, 24-Channel, 115/230 VAC, Avantor, VWR, MFLX78006-48-CH</td></tr></tbody></table>

updated protocol for the assembly and use of the minibioreactor array (mbra)

Updated Protocol for the Assembly and Use of the Minibioreactor Array (MBRA)

The human microbiome comprises diverse and dynamic microbial communities that play essential roles in host health. Understanding these communities and their responses to environmental factors is critical for advancing microbiome-based therapeutics. Traditional in vitro models for cultivating human-derived microbiota often lack scalability and require extensive technical expertise, limiting their accessibility and throughput. To address these limitations, we developed the Minibioreactor Array (MBRA) system -- a modular, single-stage, continuous-flow platform for high-throughput cultivation of microbial communities. This system enables parallel cultivation of up to 48 distinct microbial communities, supporting experimental flexibility while maintaining the stable growth of complex ecosystems. This protocol provides detailed guidance on MBRA fabrication, assembly, sterilization, and operation. The system&#39;s modular design allows for easy integration into anaerobic chambers and supports customization for a wide range of experimental applications. It has been used to study microbial responses to antibiotics, dietary compounds, and pathogen invasion, and to screen for pathogen-resistant communities. With its accessibility, scalability, and reproducibility, the MBRA represents a powerful model system for investigating microbial interactions and advancing microbiome research.

To facilitate the growth of anaerobic bacteria, such as those found in the gastrointestinal tract, the MBRA can be set up and operated inside anaerobic chambers. To demonstrate the ability to grow complex bacterial communities directly from relevant sites in the human body, a human fecal sample was prepared and grown in this system. All work was carried out in an anaerobic chamber set to 37°C, and cultures were grown in our bioreactor media, BRM37.
Fecal slurry was prepared by thawing human stool anaerobically and then mixing with phosphate-buffered saline (PBS) to a final concentration of 25% w/v. After confirming sterility, nine bioreactor chambers were inoculated each with 3 mL of the same fecal slurry. Microbial communities were grown overnight without media input or waste removal, resulting in separate batch cultures over a 16 h period. Then, the feed pumps were turned on and set to a flow rate of 1.92 mL per h. After four days of continuous flow, samples were collected from the bioreactors and analyzed for microbial community composition using 16S rRNA gene sequencing followed by denoising with Deblur and taxonomic classification with the SILVA 138 SSU database in QIIME 213. A total of 65 genera were detected across all nine replicates, but bioreactors were dominated by only 18 genera, each comprising at least 2% abundance in any of the nine replicates (Figure 5). The bioreactors showed high reproducibility such that 22 of the 65 genera were detected in all nine replicates, and an additional 17 genera were detected in at least half of the replicates. The majority of genera absent from at least one reactor (37 out of 43 genera) were rare species, each with relative abundance below 2% in bioreactors. In summary, the continuous-flow MBRA cultures supported complex, reproducible microbial communities derived from the same stool sample, even after separate batch cultures for 16 h within each bioreactor chamber.
<img alt="Bioreactor diagram showing feed and waste line trees, illustrating experimental flow system setup." class="xfigimg" src="/files/ftp_upload/68788/68788fig1.jpg" data-altupdatedat="1762509930000" data-alt="Figure 1"> 
Figure 1: The Minibioreactor Array (MBRA). Fully assembled MBRA, including labels for the feed and waste line tubing tree, the bioreactor chambers and the bioreactor strip. <a href="https://www.jove.com/files/ftp_upload/68788/68788fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="PTFE tubing preparation and straw assembly diagram for media and waste management in lab setups." class="xfigimg" src="/files/ftp_upload/68788/68788fig2.jpg" data-altupdatedat="1762509930000" data-alt="Figure 2"> 
Figure 2: PTFE etching guide and MBRA port layout. (A) Flowchart to follow for the etching of the media and waste PTFE straws. (B) Each bioreactor chamber contains a port for a media straw + threaded male luer, a waste straw + threaded male luer, and a septum + threaded male luer. <a href="https://www.jove.com/files/ftp_upload/68788/68788fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Diagram of modular pipe configurations using fittings for fluid dynamics studies and system design." class="xfigimg" src="/files/ftp_upload/68788/68788fig3.jpg" data-altupdatedat="1762509930000" data-alt="Figure 3"> 
Figure 3: MBRA feed and waste line trees. Representative images to follow in the assembly of (A) feed line tree and (B) waste line tree. <a href="https://www.jove.com/files/ftp_upload/68788/68788fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Waste containment setup; diagram shows primary to tertiary containment with connection points." class="xfigimg" src="/files/ftp_upload/68788/68788fig4.jpg" data-altupdatedat="1762509930000" data-alt="Figure 4"> 
Figure 4: Media bottle cap and waste tier system. (A) An example of an assembled Q-series bottle cap used to pull media into the MBRAs. (B) An image of the tiered waste collection system. <a href="https://www.jove.com/files/ftp_upload/68788/68788fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Relative abundance chart and diversity boxplots for microbial communities in different reactors." class="xfigimg" src="/files/ftp_upload/68788/68788fig5.jpg" data-altupdatedat="1762509930000" data-alt="Figure 5"> 
Figure 5: Relative abundance of bacterial genera. (A) Stacked bar graph shows the relative abundance of all genera that comprise at least 2% abundance in any of the samples displayed. (B) Alpha diversity metrics of Observed OTUs and Shannon Diversity between all nine bioreactor chambers. All nine bioreactor chambers were inoculated with the same fecal slurry prepared from human stool. <a href="https://www.jove.com/files/ftp_upload/68788/68788fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: MBRA components. Images and descriptions of all individual parts required to fully assemble the MBRA, along with the quantity needed for each component. <a href="https://www.jove.com/files/ftp_upload/68788/Table1.xlsx" target="_blank">Please click here to download this Table.</a>
Table 2: Troubleshooting guide. Common issues encountered with running the MBRA, along with their potential causes and recommended solutions. <a href="https://www.jove.com/files/ftp_upload/68788/Table2.xlsx" target="_blank">Please click here to download this Table.</a>
Supplementary File 1: Stl file for 3D-printing the Minibioreactor Array strips. <a href="https://www.jove.com/files/ftp_upload/68788/Supplementary%20File%201.stl" target="_blank">Please click here to download this File.</a>
Supplementary File 2: Stl file for 3D-printing the bioreactor holders used to anchor the bioreactors to the stir plates. <a href="https://www.jove.com/files/ftp_upload/68788/Supplementary%20File%202.stl" target="_blank">Please click here to download this File.</a>
Supplementary File 3.: Stl file for 3D-printing the tube holders used to organize the C-flex waste and feed tubing extending from the MBRAs. <a href="https://www.jove.com/files/ftp_upload/68788/Supplementary%20File%203.stl" target="_blank">Please click here to download this File.</a>

The Minibioreactor Array (MBRA) is a high-throughput, customizable, continuous-flow culture system that enables the cultivation of complex microbial communities, supporting parallel experiments to study microbiome dynamics, therapeutic interactions, and microbial responses to environmental factors.

The human microbiome comprises diverse and dynamic microbial communities that play essential roles in host health. Understanding these communities and ...

So our research focuses on the gut microbiome, specifically how it can be engineered to prevent the colonization of harmful pathogens. At the same time, we're also interested in understanding the ecological rules and mechanisms behind this colonization resistance. Technology, such as next generation sequencing, advanced bioinformatics, germ-free mice, and in vitro gut models, are all transforming the way we study microbial communities and their role in health and disease.One of the biggest challenges with in vitro gut models is operating them at high throughput. What we need are systems that allow for the functional interrogation of microbial communities at scale. The minibioreactor array is a continuous flow culture system that was designed to address the lack of throughput capacity in other systems.It allows us to scale up experiments while still capturing complex and reproducible microbial community behavior. To begin, ensure that the minibioreactor array strips are 3D-printed and contain six independent bioreactor chambers. Arrange all the components required for the assembly.Using a 1/4-inch 28 NF fraction tap with a T-handle tap wrench. Thread the three 1/4-inch ports in each chamber to insert fittings. After washing the chamber with water, place a 10 by 3 millimeter magnetic stir bar into each chamber and add one milliliter of distilled water.Then position a rubber washer on top of each port of the bioreactor. For each chamber, screw in one media straw threaded male lure, one waste straw threaded male lure, and one empty threaded male lure into the ports. Now insert six rubber septa onto 3/32-inch female lure barbs and fold the upper sleeve of each septum down to cover the neck.Attach these to the designated ports of each chamber. Cut C-flex tubing strips of the desired length and number. Attach a 1/8-inch female lure barb to one end and a male lure lock connector to the opposite end of each length of tubing.Then insert a 1/16-inch female lure barb into each end of the red two-stop E-lab tubing with a 1.14-millimeter inner diameter, and the orange two-stop E-lab tubing with a 0.89-millimeter inner diameter. Connect the prepared E-lab tubing to the C-flex tubing and ensure each of the six C-flex tubing lengths is connected to one red and one orange E-lab line via female lures. Next, cut the C-flex tubing to different lengths as required.Attach 1/8-inch female lure barb and a male lure lock connector to both ends of one 3-inch piece and the 12-inch piece of C-flex tubing. Attach male lure lock connectors to both ends of the remaining pieces. Assemble the waste line tree according to the 3D diagram.Attach the exposed ends of the red two-stop E-lab tubing to the terminal male lure locks on the waste line tree in ascending order based on C-flex tubing length. Then connect the 3-inch C-flex tubing with the 1/8-inch female lure barb and male lure lock connector to the top of the waste line tree. Assemble the feed line tree according to the 3D diagram.Bridge the exposed ends of the orange two-stop E-lab tubing to the terminal male lure locks on the feed line tree in ascending order based on C-flex tubing length, and attach the 12-inch C-flex tubing to the top of the feed line tree. Attach the variable length C-flex tubing at the end of the feed line tree to the bioreactor, arranging them in ascending order from the shortest line on the left to the longest on the right. Attach the variable length C-flex tubing at the end of the waste line tree to the bioreactor strip in descending order with the longest line on the left and the shortest on the right to to accommodate pump placement.Bundle all C-flex feed lines together on the left side of the strip and secure them with a twist tie. Form a loop with the orange two-stop E-lab tubing between the C-flex lines and secure the loop using autoclave tape, and repeat the process for the red two-stop E-lab tubing on the waste side of the bioreactor strip. Cover the female lure at the end of the waste and feed line trees with foil to prevent contamination.Loosen the male threaded lures with septa on each bioreactor chamber to allow steam to escape during autoclaving. After placing the assembly into an autoclave bin, stretch out the feed and waste line trees into separate bins adjacent to the one containing the MBRA strips. To attach the system to the pumps, remove the autoclave tape securing the E-lab tubing for both waste and feed lines, and untie the bundles of C-flex tubing.Position the MBRA between the two pumps on top of the stir plate. Clamp it down using the 3D printed holders and align it with the marked stirring positions on the plate. Now attach the feed line E-lab tubing to the peristaltic pump cartridges and position the tubing stops into the cartridge slots.Repeat the process for the waste line E-lab tubing on the pump located to the right of the stir plate. Then lock the peristaltic pump cartridges into the pump. Arrange the C-flex tubing neatly using the 3D printed tube holders.Connect the end of the waste line tree to the tubing connected to the waste bottles. Next, attach the female lure on the feed line entry tubing to the male connector on the 12-inch tube from the media bottle cap. Turn on both pumps to start media flow and ensure both pumps are set to clockwise rotation when waste is positioned to the right of the pumps.Observe the droplet size and cadence in each bioreactor chamber. If any variability or abnormality is noticed, replace the orange two-stop E-lab tubing connected to the affected chamber to reduce flow rate variation. Once the chambers are full, shut off both pumps and allow the bioreactors to sit for 24 to 48 hours to check for contamination before starting the experiment.Switch the media input to a one liter container of 10%bleach in deionized water and increase the flow rate on both pumps to maximum to displace the contents of the bioreactor chambers with bleach. Once the chambers are clear of media, invert the MBRA to disinfect above the fill line for five minutes. After five minutes, right the system and wait an additional five minutes for sterilization.After the chambers have been cleared of media and have been sterilized for 10 minutes, replace the bleach with one liter of deionized water and flush the system until the water has passed through. Then disconnect the bioreactor E-lab tubing from the pumps and remove the MBRAs. Remove the used septa from the bioreactor and drain each chamber until only one milliliter of water remains.Replace the septa, the orange two-stop E-lab tubing, and autoclave the completely assembled strip as demonstrated previously. After three cycles of reuse, follow these steps. A human fecal sample was prepared and grown in the MBRA system.After four days of continuous flow, the microbial community in all nine bioreactors was dominated by 18 bacterial genera, each comprising at least 2%of relative abundance in any replicate. 22 out of the 65 detected genera were present in all nine bioreactor replicates, demonstrating high reproducibility. Alpha diversity analysis showed minimal variation between replicates in both the observed operational taxonomic units and Shannon Diversity Index.

updated-protocol-for-assembly-use-minibioreactor-array

Research

JoVE Journal

Biology

Source: Hemmerich, J., et al. Protocol for Optimization of Heterologous Protein Production Using Automated Microbioreactor Technology. J. Vis. Exp. (2017).This video demonstrates the use of an automated microbioreactor to monitor bacterial growth and fluorescent protein secretion in response to varying media formulations. A genetically modified bacterium expressing a secretory fluorescent protein is cultured under antibiotic selection and inoculated into nutrient-varied wells. Real-time optical...

Monitoring Bacterial Growth in Distinct Media Formulations with an Automated Microbioreactor

In this protocol the fabrication, setup and basic operation of a microfluidic picoliter bioreactor (PLBR) for single-cell analysis of prokaryotic microorganisms is introduced. Industrially relevant microorganisms were analyzed as proof of principle allowing insights into growth rate, morphology, and phenotypic heterogeneity over certain time periods, hardly possible with conventional methods.

Microfluidic Picoliter Bioreactor for Microbial Single-cell Analysis: Fabrication, System Setup, and Operation

An amplification microarray combines asymmetric PCR amplification and microarray hybridization into a single chamber, which significantly streamlines microarray workflow for the end user. Simplifying microarray workflow is a necessary first step for creating microarray-based diagnostics that can be routinely used in lower-resource environments.

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

Using a pneumatic bioreactor, we demonstrate the assembly, operation, and performance of this single-use bioreactor system for the growth of mammalian cells.

Cultivation of Mammalian Cells Using a Single-use Pneumatic Bioreactor System

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Here, the HDBR is successfully applied in a photobioreactor (PBR) configuration for the study of nitrogen metabolism by a mixed high density algal community.

A Novel Bioreactor for High Density Cultivation of Diverse Microbial Communities

In this two-part study, a biological actuator was developed using highly flexible polydimethylsiloxane (PDMS) cantilevers and living muscle cells (cardiomyocytes), and characterized. The biological actuator was incorporated with a base made of modified PDMS materials to build a self-stabilizing, swimming biorobot.

Cardiac Muscle-cell Based Actuator and Self-stabilizing Biorobot - PART 1

The development of microbial communities depends on a combination of factors, including environmental architecture, member abundance, traits, and interactions. This protocol describes a synthetic, microfabricated environment for the simultaneous tracking of thousands of communities contained in femtoliter wells, where key factors such as niche size and confinement can be approximated.

Assembly and Tracking of Microbial Community Development within a Microwell Array Platform

This manuscript describes a generic approach for tailor-made design of microbial cultivation media. This is enabled by an iterative workflow combining Kriging-based experimental design and microbioreactor technology for sufficient cultivation throughput, which is supported by lab robotics to increase reliability and speed in liquid handling media preparation.

Generic Protocol for Optimization of Heterologous Protein Production Using Automated Microbioreactor Technology

Here, we present a detailed procedure to run a Design of Experiment in an automated micro-bioreactor followed by cell harvest and protein quantification using a Protein A column.

Process Optimization using High Throughput Automated Micro-Bioreactors in Chinese Hamster Ovary Cell Cultivation

This protocol details the assembly of mini-bioreactor arrays to be utilized for continuous flow culture of complex fecal communities under anaerobic conditions. We also discuss the methods for assembly, inoculation, and sampling of the reactors for further analysis.

Bioreactor Assembly for Continuous Culture of Complex Fecal Communities

A protocol is described for microcarrier-microbioreactor production of anchorage-dependent cells as potential cell-based therapies. Targeting a use case of acute respiratory distress syndrome, comparable cell yield and favorable gene expression relative to flask-based process conditions are illustrated. This protocol is extensible to other anchorage-dependent cells and target indications.

Microbioreactor-Based Production of Anchorage-Dependent Mesenchymal Stromal Cells Primed for Acute Respiratory Distress Syndrome

Scopri l'assemblaggio e l'uso dell'array di minibioreattori (MBRA) per studiare la dinamica delle comunità microbiche intestinali ad alto rendimento. Questo metodo consente di analizzare i meccanismi di resistenza alla colonizzazione dei patogeni.

Updated Protocol for the Assembly and Use of the Minibioreactor Array (MBRA)

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