Recent demand for renewable and sustainable forms of portable chemical energy have led to renewed interest in and development of Aquatic Microbial Oxygenic Photosynthetic organisms (AMOPs) as platforms for biofuel production. The potential use of AMOPs in “cell factory” applications, in which biofuels or fuel precursors are excreted from cells without the need to destructively harvest the biomass is particularly appealing. We have examined existing analyses of the energy efficiency and net greenhouse gas release of AMOP fuel production, developed a fair comparison of these studies, and provided revised projections of and targets for improvement of the AMOP fuel process chain. We have developed and optimized a targeted LC-MS2 analytical method to monitor the intracellular metabolome of the model marine cyanobacterial AMOP Synechococcus sp. PCC 7002 under fuel-producing conditions. We have applied this methodology to characterize and identify bottlenecks in auto-fermentation, analyzing the wild-type strain alongside several genetic and environmental perturbations. We have gained insight into the limitations of fermentative metabolism in this organism and identified key targets for enhancement of the rate and yield of the production of biofuels such as hydrogen.
In Chapter One, we describe a meta-analysis of several recent Life Cycle Analysis (LCA) studies in which the net energy efficiency and Global Warming Potential (GWP) of AMOP biofuels are estimated. We have developed holistic models of Destructive Harvesting and Milking approaches to fuel production and performed a direct comparison of the values and assumptions employed by the various studies. We subsequently put forth new meta-data based scenarios for both models and report net positive energy production and GHG mitigation potential in the majority of cases. Lastly, we see fewer barriers towards enhanced efficiency in the Milking model alongside greater potential for long-term viability in the wake of probable responses to global climate change.
In Chapter Two, we report the development of a method for the chemical isolation and tandem Liquid Chromatography – Mass Spectrometry (LC-MS 2) quantification of a targeted subset of internal metabolites from Synechococcus, a model marine cyanobacterium capable of producing valuable products such as lactate, acetate, and hydrogen. We describe the selection of target compounds, requirements for and optimization of mass spectral detection channels, screening and optimization of chromatography, and development of a complete, rapid, and stable sampling protocol. We identify and resolve several key factors influencing the separation by reversed-phase ion pairing (RIP) chromatography, specifically the hydrophobicity of the sample matrix and sensitivity to mobile phase acidity. We apply this methodology to an initial analysis of auto-fermentative metabolism in Synechococcus, for which intracellular levels of 25 metabolites were monitored over 48 hours, including intermediates in central carbon metabolism together with those involved in the cellular energy charge and redox poise. Upon removal the alternative reductant sink nitrate, auto-fermentation induces a rise in the intracellular pyridine nucleotide redox poise that is specific to NAD(H) alongside a gradual decline in the adenylate energy charge, corroborating previous observations using alternative methods of analysis.
In Chapter three, this LC-MS2 method is applied to analyze the primary constraints on auto-fermentative metabolism in Synechococcus , a “cell factory” state in which terminal products are excreted from the cell and into the environment. The time-dependent changes in intracellular intermediates point to a primary bottleneck at GAPDH (1.2.1.12), the sole nucleotide reduction step in the Embden Meyerhof Parnas (EMP) pathway, corroborated by the measured rise in intracellular NADH and concomitant drop in NAD+. A disequilibrium between these and the NADP(H) pools indicates low transhydrogenase activity, supported by transcriptional and biochemical data. Oxidant specificity during auto-fermentation is demonstrated by a shift in catabolism from the EMP to the oxidative pentose phosphate (OPP) pathway in the presence of the NADPH-specific reductant sink nitrate. Global changes in central carbon metabolism and affiliated energy carriers in the presence of glycerol provide evidence of increased upper glycolytic backup and activation of the alternate OPP pathway. Lastly, an overlay of cDNA sequencing-based transcriptional data onto the metabolomic model suggests that gene expression of EMP enzymes exacerbates the constraint of auto-fermentative flux at GAPDH. These results highlight crucial targets for increasing the rate of carbohydrate catabolism towards the “overnight” target needed for biofuel application and improving the yields of valuable fermentative products, such as H2, ethanol, and lactate.
