The Mini Colon Model: a benchtop multi-bioreactor system

This tech spec was submitted by Jin Zijie as part of the University Technology Exposure Program.

Problem / Solution

With the use of in vitro fermentation systems, it is made possible to study the gut microbial communities while precisely controlling several physiological parameters and separating confounding elements from the human host. However, conventional systems like SHIME and Robogut are seen as having a big footprint, lacking multiplexing, and having low experimental throughput. Options to get around these limits, like the Mini Bioreactor Array System, are expensive pieces of specialized equipment that can't be used by many facilities.

The Mini Colon Model (MiCoMo), a low-cost benchtop multi-bioreactor device, can replicate the physiologically accurate conditions of the human colon. The MiCoMo is a tiny, anaerobic, pH-controlled, and optimized bioreactor system. Additionally, this apparatus has triplicate bioreactors that operate separately from an anaerobic chamber, as well as automated pH, temperature, and fluidic control. The device's high throughput and multiplexing capabilities make it an accessible technology for researching gut microbial communities.

Design

Three single-stage reactors with a working volume of 30 mL and a capacity of 55 mL are installed in the small colon model (MiCoMo). Additionally, each reactor is outfitted with a gas sparging line for N2 flushing to maintain anoxia as well as an acid/base adjustment and fluidic transfer tubing with Luer-lock connectors. The anaerobic reactors manage to maintain 37°C in a water bath and can be housed in a biosafety cabinet during the operation of the MiCoMo to avoid potential contamination. Additionally, for all trials in the study, the small colon model runs on a 4-hour feed cycle. Additionally, the feed cycle results in a 30-hour total reactor turnover time.

Media Preparation

A Modified Gifu Anaerobic Medium (mGAM) serves as the media type for the MiCoMo. This media is made by dissolving 41.7 g of powder in 1 L of distilled water, and it is autoclaved at 121 °C for 30 minutes to sterilize it. Additionally, antifoam 204 at 0.01 percent is added to the media to prevent foam from forming during the experiment.

MiCoMo sampling and operation

To prepare for fecal injection, MiCoMo reactors are sterilized by being incubated in 70% ethanol for 1 hour. After that, the system is put together in a biosafety cabinet, and 30 minutes are spent running 10 percent bleach through all of the reactors' piping connections. After that, the system is flushed for 5 minutes with sterile MilliQ water. The pH probes were then sanitized by immersing them in 10% bleach for an hour. To increase oxygen scavenging and induce anoxia after fecal inoculation, 1 mL of a 40 gL-1 L-cysteine solution was added to 99 mL of sterile mGAM with antifoam. This extra L-cysteine was only added to the media used for the initial seeding; feed media was not used for the rest of the experiment.

A pH control system and nitrogen flushing are then carried out. Before reactors are seeded with feces through their seeding ports, the pH set point is maintained for at least 30 minutes. For all trials, a pH of 6.7 with a +/- 0.1 tolerance is maintained. After inoculation, a 4-hour feed cycle is also started, with each cycle involving the removal of 4 mL (13 percent) of the reactor content and the addition of 4.5 mL of fresh media. Additionally, the extra media is utilized to make up for the daily 3ml4mL14,000gFurther

Results

The MicoMo can handle the rapid stabilization of intricate microbial communities and maintain volunteer volunteers' microbial diversity for roughly three to five days. Additionally, due to its compact design, low cost, and simplicity of manufacture, it is simple to replicate across laboratories, allowing for a high throughput analysis of the impact of different perturbations on distinct gut microbial populations.