Nitrogen is an essential component of many cellular constituents such as proteins and nucleic acids. Although the Earth's atmosphere is nearly 79 % nitrogen, free gaseous nitrogen (N₂) cannot be utilized by most living organisms. The N₂ molecule is chemically very stable and must be “fixed” into compounds like nitrates or ammonia before it can be assimilated.

Nitrogen fixation is performed naturally by a number of different prokaryotes, including both free-living bacteria and symbionts (Rhizobia) that form associations with leguminous plants. Rhizobia form colonies inside specialized nodules in the roots, producing fixed nitrogen in exchange for the carbon source (e.g. sugars) provided by the plants. Nitrogen fixation is carried out under microaerobic conditions at atmospheric pressure and temperature via an iron-rich enzyme, nitrogenase, a remarkable feat that synthetic catalysts have been unable to match.

N₂ + 8 H+ 16 ATP -> 2NH₃ + H₂ + 16 ADP

In contrast, industrial ammonia synthesis is performed through the famous Haber-Bosch process, an energy-intensive reaction occurring at 400-650 °C and 200-400 atm in the presence of an iron based catalyst.

N₂(g) + 3H₂(g) -> 2NH₃(g)

5 % of the natural gas consumed in the U.S. is expended in producing H₂ for ammonia synthesis. In certain third world countries with agricultural economies, up to 40 % of fossil fuel expenditures are devoted to production of ammonia and nitrates for chemical fertilizers.

Unlike the Haber-Bosch process, biological nitrogen fixation actually produces H₂ gas, a valuable by-product, rather than consuming it, and the required energy inputs (carbon source) can be obtained from biomass sources rather than fossil fuels. Thus, the use of microbes for ammonia synthesis could potentially reduce dependence on fossil fuels, while providing a source of hydrogen gas from renewable resources such as plant-derived sugars. However, it is not clear whether the biological process can be harnessed to produce ammonia/hydrogen in an economically feasible manner, or if the process might be limited by low reaction rates, scale-up issues, or contamination problems.

We are therefore designing a laboratory-scale, continuous-flow, packed bed bioreactor system to test the feasibility of biological ammonia synthesis in a continuous flow reactor system for the first time. Our preliminary work has examined both free-living nitrogen fixers (e.g. Azotobacter or certain Clostridia) and Rhizobia. We are using a commercial benchtop fermentor to grow and maintain a high density of bacteria in a controlled environment. Bacteria or bacteroids are supported by a porous polymer hydrogel scaffold in a flow-through, aqueous environment that provides the microbes with a continuous flow of nutrients, while removing waste products and ammonia.

Convincing (usually aerobic) nitrogen fixing bacteria to conduct ammonia synthesis outside of their natural environment presents a significant materials research challenge, however, due to the need to regulate oxygen diffusion. The bacteria require at least microaerobic O₂ concentrations to survive, but excess oxygen irreversibly deactivates nitrogenases, the key enzymes involved in N₂ fixation. In nature, legume root nodules help regulate delivery of oxygen at levels that will be sufficient to carry out metabolic activities, but low enough to maintain active nitrogenase. Thus, to encourage nitrogen fixation in an artificial environment, a biomimetic scaffold material is needed that approximates the root nodule environment, providing a surface for attachment of the bacteria, while helping to regulate O₂ permeation. We are developing porous, water-swollen polymer networks (hydrogels) that meet these requirements and preserve the possibility of scale-up at a later stage.

Our presentation will discuss recent progress in reactor design and development of porous, autoclavable hydrogel scaffolds. Recent progress in achieving support of large, active colonies of various nitrogen fixers will also be discussed.