Biohydrogen from Cyanobacteria & Microalgae

Robust Microalgal Production Strains for High Yield Growth on Fossil Flue Gas

Algae are the most productive photosynthetic organisms at solar energy conversion, by far. Many algal species are known to accumulate high concentrations of oil (Triacylglycerides, TAGs) up to 50% or more of their cellular dry weight. Unicellular marine microalga Nannochloropsis Oceanica CCMP 1779 is a prospective production strain for CO2 mitigation and biofuel production. By collaborating with Dr. Christoph Benning, we generate potential flue gas tolerant Nannochloropsis CCMP1779 strains by random mutagenesis combined with high throughput screening as well as targeted genetic engineering of selected genes involved in fatty acid biosynthesis, TAG assembly and TAG catabolism pathways.

 

 

All oxygenic phototrophs extract electrons and protons from water and use them to reduce NADP+ for the use as energy sources in metabolism such as CO2 fixation via the Calvin cycle and other pathways. However, some microbial oxygenic phototrophs (cyanobacteria and microalgae) can transiently produce H2 gas under anaerobic conditions via proton reduction catalyzed by an enzyme, hydrogenase. Cyanobacteria, unlike microalgae, prefer a dark auto-fermentation process that follows the light-dependent photosynthetic stage to produce hydrogen.

 

All oxygenic phototrophs extract electrons and protons from water and use them to reduce NADP+ for the use as energy sources in metabolism such as CO2 fixation via the Calvin cycle and other pathways. However, some microbial oxygenic phototrophs (cyanobacteria and microalgae) can transiently produce H2 gas under anaerobic conditions via proton reduction catalyzed by an enzyme, hydrogenase. Cyanobacteria, unlike microalgae, prefer a dark auto-fermentation process that follows the light-dependent photosynthetic stage to produce hydrogen.

 

The anaerobicH2 producing metabolism breaks down the stored glycogen into a C3 intermediate carbohydrate that enters either the oxidative pentose phosphate pathway or the glycolysis pathway (see Figure 1). In the OPP pathway they are stripped of hydrogen to reduce NAD(P)+with release of CO2as byproduct, while in the glycolysis pathway the C3 intermediate is only partially stripped of hydrogen to reduce NAD+ with release of organic acids. The reductant yield differs 3 fold for OPP vs glycolysis, and entry into these pathways is highly regulated. The reductant generated may be used as a substrate by hydrogenase to produce hydrogen gas.

 

 

 

Previous work from our lab has shown that H2 production is not limited by the enzyme but by substrate availability {Ananyev, 2008}. Therefore the challenge in engineering cyanobacteria as hydrogen producing factories is to maximize the reductant availability to hydrogenase with minimum effect on the cell growth and viability. With this in mind, our first stage of the project was to develop quantitative experimental tools for simultaneous measurements of multiple intracellular metabolites {Bennette, 2011},{Carrieri, 2009},{Ananyev, 2008},{Bennette, 2008}so as to delineate the H2metabolic network{Skizim, 2012} in a variety of cyanobacteria. Using this approach, our team applies the following strategies to increase the flux of carbohydrate catabolism to H2in cyanobacteria (Figure 2):

 

1) Eliminating pathways that compete with hydrogenase for substrate: Multiple auto-fermentative pathways consume NADH thus limiting the substrate for hydrogenase. We have shown that the deletion of these pathways by genetic knock-outs especially in genes for lactate dehydrogenase and pyruvate-ferredoxinoxidoreductase in Synechococcussp. 7002 led to almost a 5-fold increase in hydrogen production {McNeely, 2010},{McNeely, 2011}. 

2) Altering the carbon decision tree to enhance reductant producing pathways: We are involved in understanding the effect of the form and availability of the glycolytic substrate on the rate of catabolism and thus on hydrogen production. In salt tolerant strains environmental stresses that increase in carbohydrate accumulation have been applied that increased catabolism by 8 fold {Carrieri, 2011} or accelerate the rate and and increase the yield of H2 production by milking, or by reverse fermentation .Synechococcussp 7002 mutants altered in glycogen accumulation demonstrate the importance of polymeric sugars in enhancing glycolysis {Guerra, 2012, submitted},{Xu,2012, submitted} 

3) Modifying metabolic choke-points that regulate the rate of carbohydrate catabolism: The rate of pyridine nucleotide reductant generation under dark auto-fermentative conditions is limited by the rate of catabolism of sugar and inhibited by the accumulation of glycolytic intermediates that cause feedback inhibition {McNeely, et al; Bennette et al, in preparation}. Overexpression and knock-out mutants have been designed to either circumvent or remove these choke-points and thus enhance reductant availability. Special focus has been placed on glycolytic enzymes like GAP, ADK etc., {Kumar, in preparation}. 

4) Modifying the NiFe-bidirectional hydrogenase (Hox) to increase the rate of H2 production: Providing adequate Ni2+for assembly of the NiFe-hydrogenase without photo-inactivation/bleaching is a special problem in oxygenic phototrophs that we have overcome . Protons are co-substrates with NADH for H2 production by hox hydrogenases. We are genetically engineering a hox hydrogenase to increase the local proton availability through addition of buffer binding domains and allosteric regulation sites for activation of H2 production.