The title of our MURI project is “Renewable Bio-solar Hydrogen Production from Robust Oxygenic Phototrophs”. The project engages 8 PIs from different institutions working with 26 PhD or doctoral candidates and about 20 undergraduate trainees. We call the group the BioSolarH2 team. http://www.princeton.edu/~biosolar/
The project aims to coax unicellular microbes called cyanobacteria and algae to serve as “cell factories” to make hydrogen gas by diverting some of their internal energy storage molecules (C molecules: starch, glycogen and sugars and N molecules). The microbes are photosynthetic, so they use solar energy to make these molecules from readily available environmental nutrients like CO2, water, and nitrate, while releasing oxygen gas. After their utility as cell factories for producing H2 gas is spent, these microbes represent a major source of biomass, which is completely devoid of recalcitrant cellulose and lignin. This biomass can be fermented more easily than plant biomass using other (non-photosynthetic) organisms to produce more H2 or liquid fuels.
Algae and cyanobacteria use either of two mechanisms to make H2, but in both cases they can only do so in the absence (partial or complete) of O2 gas. This is one of the key limitations the team is working to alleviate. Another is mining the enormous diversity of photosynthetic microbes which exists in nature - differing in ecological habitat and biosynthesis reactions - to identify promising candidates. There is an extensive screening program in place supported by bioprospecting in the field. Several lead candidates have been identified from the Great Salt Lake, thermal springs, volcanic and marine sources.
The algae as a class seem to prefer a direct process in which sunlight, which normally carries out photosynthetic charge separation (converting water O2 + electrons + protons) occurs as usual, but the photo-products are diverted into H2 production via a hydrogenase, the major hydrogen-producing class of enzymes. Cyanobacteria as a class seem to prefer an indirect dark process that follows the light-dependent photosynthetic stage. This process converts glycogen (strong C-H bonds) into smaller C molecules by a well known process called glycolysis, and ultimately some of these molecules are oxidized fully into CO2 by another well studied process called fermentation. These processes produce an intracellular form of hydrogen (called NADH or NADPH) and the energy-rich molecule ATP (which can be converted to protons). These are combined in the cells via a hydrogenase to make H2 gas. The challenge here is that all three metabolic pathways that are needed: growth by photosynthesis, respiration of glycogen and anaerobic fermentation of C intermediates, occurs in a single multi-functional organism. So the coordination/interference of these reactions and their inherent activities need to be optimized in order to attain practical yields of H2. The BioSolarH2 team is adopting multiples strategies to accomplish this. One involves the application of environmental stresses to accelerate the rate of the slow fermentation process so as to better match the natural diurnal cycle of the sun. For example, cyanobacteria that are stressed by osmotic shock via salt dilution can pump out H2 at 20-fold faster rates. Another approach is to genetically alter or knockout the genes for enzymes that compete with the desired pathways to hydrogenase-dependent H2 production. This approach has just begun, but the first result has demonstrated a 2-fold increase in the yield of H2 production. Multiple other pathways are under investigation. A molecular based approach is also under investigation using computational chemistry to model the detailed atomic pathways that hydrogenases appear to use to produce H2. This knowledge may guide the construction of mutant hydrogenases with enhanced properties.
In order to monitor the inner workings of these microbes, the team has designed and fabricated extremely powerful fluorescence and electrochemical tools and bioreactors to measure the concentration of intermediates and end products for these competing reactions. One of the key results from this project is the first evidence for resolution of two temporal phases of H2 production within the cells, arising from precursors produced by different metabolic pathways. The team is also applying powerful mass spectrometric and NMR methods for simultaneous identification of large numbers of metabolites, as well as DNA microarrays to monitor the expression of genes that respond to H 2 producing conditions. The project is converging on a holistic picture of the cellular machinery needed for more efficient biomass production and energy transformation in cyanobacteria and algae.
Figure legend: Graduate students from Princeton University, Kelsey McNeely, Nick Skizim and Damian Carrieri, transferring cyanobacteria cultures.