The aspiration for producing algal biofuel is motivated by the desire to replace conventional petroleum fuels, produce fuels domestically, and reduce greenhouse gas emissions. Although, in theory, algae have the potential to produce a large amount of petroleum fuel substitutes and capture carbon emissions, in practice, profitable algal biofuel production has proven quite challenging. This dissertation characterizes the production pathways for producing petroleum fuel substitutes from algae and evaluates constraints on algal biofuel production. Chapter 8 provides a summary of the entire dissertation. The first chapter provides a framework for reporting the production of renewable diesel from algae in a consistent way by using data that are specific and by presenting information with relevant metrics. The second chapter presents a review of analytical tools (i.e., microscopy, spectroscopy, and chromatography) that can be used to analyze the structure and composition of intermediate products in an algal biofuel production pathway. In chapters 3 through 6, the energy return on investment, water intensity, and financial return on investment are presented for three cases: 1) an Experimental Case in which data were measured during five batches of algal biocrude production with a combined processed volume of about 7600 L, 2) a hypothetical Reduced Case that assumes the same energy output as the Experimental Case, with reduced energy and material inputs, and 3) a Highly Productive Case that assumes higher energy outputs than the Experimental Case, with reduced energy and material inputs, similar to the Reduced Case. For all three cases, the second-order energy return on investment was determined to be significantly less than 1, which means that all three cases are energy negative. The water intensity (consumption and withdrawal) for all cases was determined to be much greater than that of conventional petroleum fuels and biofuels produced from non-irrigated crops. The financial return on investment was also found to be significantly less than 1 for all cases, indicating production would be unprofitable. Additionally, it was determined that large-scale algal biofuel production would be constrained by the availability of critical energy and material inputs (e.g., nitrogen and carbon dioxide). The final part of this dissertation presents a first-principles thermodynamic analysis that represents an initial attempt at characterizing the thermodynamic limits for algal biofuel production. In that analysis, the energy, entropy, and exergy is calculated for each intermediate product in the algal biofuel production pathway considered here. Based on the results presented in this body of work, game-changing technology and biotechnology developments are needed for sustainable and profitable algal biofuel production. / text