Methanogens

Why use microfluidics?

Despite its many fluid mechanics applications, the greatest potential for microfluidics lies, in fact, in biology and medical science. Since microfluidic networks are biocompatible and are on the same length scale as most cells, they provide an ideal environment for creating microcultures and performing assays. The microorganism we are currently studying is the waste-water methanogen, methanosaeta concilii. Conventional studies on the behavior of methanogens (methane-producing, obligate anaerobic archaea) have been conducted in large serum bottles or reactors in order to maintain an anoxic environment. Such systems are limited since the growth of the methanogens cannot be studied in situ. By using microfluidics to culture methanogens, we have the ability to perform direct microscopy without sampling disturbance. We have developed a micro-bioreactor (uBR) with a total system volume of 5 micro-liters. The uBR is contained inside an anaerobic chamber designed to be placed directly onto a microscope stage, while maintaining a N2/CO2 gas mixture.

Methanosaeta Concilii growing inside a microfluidic network

What are methanogens?

Despite being unable to tolerate the presence of any oxygen, methanogens are surprisingly found in a wide variety of environments. They are used in waste-water treatment, grow at the bottom of lakes and ponds and even grow inside our intestinal tracts. With regards to the methanogen used in our study, methanosaeta concilii, a single cell is generally around 1 micron in size. These single cells often link together into larger filaments that can be several hundred microns in length. These filaments will then sometimes wrap around one another into even larger bundles or clumps as shown in the picture above. These methanogens play a key role in anaerobic waste digestors used on farms throughout the world. Currently, however, these digestors have up to a 70% failure rate. Obviously there is still a great deal that can be done to optimize their performance.

Schematic diagram of the micro-bioreactor

How do we know our system is anaerobic? A special indicator called resazurin was added into the methanogen media. In a completely reduced environment (one with no oxygen) resazurin is completely colorless. Once it comes into contact with oxygen, however, it turns a bright red or pinkish color. All of the effluent in our system is completely colorless, meaning no oxygen is present and the methanogens have a hospitable environment to grow in.

The picture on the left was taken while the reactor was sealed and being maintained under an overpressure. The picture on the right was taken shortly after the chamber was opened. As can be Once the media comes into contact with oxygen, it's color becomes bright pink.

Results

Shear Stress

In a first experiment, methanogens were cultured for up to 3 months inside channels of varying width (shown to the right). The varying channels widths created varying fluid velocities (and thus varying shear stresses). This allowed for direct study of the behavior and responses of the anaerobe to varying shear-stresses. Based on these growth conditions, an optimum shear level of approximately 6-8 uPa was found. At shear stresses above this range, the methanogens were pulled off of the surfaces. Below this range, media turnover was believed to be too low, resulting in the cells receiving too little nutrition. Based on this initial study, we knew what flow-rates to use for all our future experiments.

Microfluidic geometry used for the shear studies

pH and Ammonia

Subsequent research examined optimal media properties for the growth of methanogens. In these studies we utilized the microfluidic gradient design shown to the left. The wavy portion of the network works to mix the two inlet fluids. Since the flow is laminar, there will be no convective mixing in the outlet, resulting in clearly defined bands of varying concentration of the two fluids (below).

Outlet of the gradient network. Green dye was pumped into the left inlet and water was run into the right.

Microfluidic gradient pattern

Two variables were examined, pH and ammonia levels. In the first study, pH levels ranging from 5.5 to 8.5 were examined and an optimum of approximately 7.6 was found (below). In a second study ammonia levels ranging from 250 to 2500 mg N-NH4/L were used. Ammonia levels above approximately 750 mg N-NH4/L were found to inhibit methanogenic growth.

Results of the pH gradient studies (red and blue triangles) overlayed onto previosuly reported data based on methane production of large-scale reactors at different pH levels (green circles). Dashed lines represent Gaussian curve fittings of the data.

Our current research

Ongoing research involves the optimization of our fluorescent in situ hybridization (FISH) procedure inside the microchannels, as well as examining additional applications of this technology.

Bacterial contamination in the reactor stained with DAPI (blue) and a general bacterial probe (red).

Useful Links

Steinhaus, B., Garcia, M. L., Shen, A.Q. and Angenent, L.T. "A Portable Anaerobic Micro-Bioreactor Reveals the Optimum Growth Conditions for the Methanogen Methanosaeta concilii" Applied and Environmental Microbiology 73(5): 1653-1658

Methanosaeta concilii growth media

Oregon Collection of Methanogens

Contact Info

Name:

Benjamin Steinhaus

Email:

bcsteinhaus@yahoo.com