A Portable Anaerobic Microbioreactor Reveals Optimum Growth Conditions for the Methanogen Methanosaeta concilii

Abstract

Conventional studies of the optimum growth conditions for methanogens (methane-producing, obligate anaerobic archaea) are typically conducted with serum bottles or bioreactors. The use of microfluidics to culture methanogens allows direct microscopic observations of the time-integrated response of growth. Here, we developed a microbioreactor (BR) with ϳ1-l microchannels to study some optimum growth conditions for the methanogen Methanosaeta concilii. The BR is contained in an anaerobic chamber specifically designed to place it directly onto an inverted light microscope stage while maintaining a N 2 -CO 2 environment. The methanogen was cultured for months inside microchannels of different widths. Channel width was manipulated to create various fluid velocities, allowing the direct study of the behavior and responses of M. concilii to various shear stresses and revealing an optimum shear level of ϳ20 to 35 Pa. Gradients in a single microchannel were then used to find an optimum pH level of 7.6 and an optimum total NH 4 -N concentration of less than 1,100 mg/liter (<47 mg/liter as free NH 3 -N) for M. concilii under conditions of the previously determined ideal shear stress and pH and at a temperature of 35°C.Microfluidic networks have recently gained importance for their wide variety of microbial applications. For example, microchannels were used by DiLuzio et al. (11) to examine the swimming behavior of Escherichia coli. DiLuzio et al. showed that E. coli sensed the presence of channel walls at distances of up to 10 m. Balagaddé et al. (5) built a microfluidic bioreactor containing a feedback control loop, which was able to correlate sustained oscillation in the cellular density of planktonic E. coli with morphological changes. The microfluidic networks used in these studies allowed researchers to directly observe the responses of microbial cells to various stimuli and provided new and unique insights into the growth and behavior of these cells. The use of microfluidics has become practicable because of the development of an inexpensive, biocompatible, and transparent but readily diffusive polymeric material (i.e., polydimethylsiloxane [PDMS]), which is used to construct micron-scale fluid networks in virtually any two-dimensional configuration (12, 23). Due to the extensive gas permeability of PDMS and the elevated cost of nondiffusive materials to construct microchannels, the application of microfluidics to the study of the growth and behavior of anaerobic microorganisms has been hindered.Conventional studies of the behavior of anaerobes, their responses to various stimuli, and their attachment have been performed with medium bottles or bioreactors ranging anywhere from several milliliters to several liters in size (2, 3, 6, 24). These systems serve to provide the anaerobic conditions necessary for growth. They do not, however, allow for any type of direct observation (without sampling disturbance) of microbe behavior, morphology, or the ability to attach to a matrix during growth. By utilizing microfluidics, ...

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