It has been long known that algae have the potential to produce a diverse range of metabolic products including lipid and starch, which could be utilized as a fuel feedstock. Despite the capacity of algae to synthesize and store large amounts of lipids and starch, algae are not currently a commercially viable feedstock for biofuel. The metabolite storage in algae can depend on the availability of nutrients such that nutrient starvation can boost the storage of lipid and carbohydrate. These nutrient-status-induced changes in lipid and starch are underpinned by altered expression of several metabolite-related genes. However, many aspects of fatty acid and carbohydrate biosynthesis are not well understood. Furthermore, the genetic regulators of nutrient starvation-induced carbohydrate and lipid accumulation are unknown in microalgae. Therefore, this PhD focused on screening cultivation conditions, in particular Phosphorus (P) and Nitrogen (N) limited conditions that induce metabolic changes, evaluated a rapid microalgal screening method, which was used to identify putative metabolism regulators, and characterized in detail the role of one P-starvation regulator, called PSR1 (Phosphorus starvation response 1). For establishing suitable culture conditions, the microalga Chlamydomonas reinhardtii was cultured in five different P and N-limited conditions and screened for metabolic changes using Fourier transform infrared spectroscopy (FT-IR) at different phases of growth. The FT-IR spectral changes were visualized by multivariate statistical tools such as principal component analysis (PCA) and principal component-discriminant function analysis (PC-DFA). Clear clustering based on nutrient availability and metabolic changes demonstrates the potential and sensitivity of FT-IR in screening multiple culture conditions. The potential of FT-IR was further tested by screening mutant strains of C. reinhardtii that were defective in response to nutrient starvation. Nine lines with mutation in one or more of the PSR1, SNRK2.1 or SNRK2.2 genes and a wild type were screened by FT-IR for P and N starvation-induced metabolic changes. PCA, PC-DFA and predictive partial least squares discriminant analysis (PLS-DA) of FT-IR spectra, clearly distinguished wild type from mutant strains and clustered mutants with similar genetic backgrounds, demonstrating the potential of FT-IR to detect and differentiate specific genetic traits. The changes in lipid and carbohydrate profile under nutrient stress and in the different strains were validated by biochemical analysis and liquid chromatography-mass spectrometry (LC-MS).This thesis demonstrated that PSR1 is an important regulator of neutral lipid and starch biosynthesis. Transcriptomic analysis on wild type and psr1 mutant under P-starvation was performed to identify transcripts induced by P-starvation that were mis-regulated in psr1. Mainly transcripts encoding starch and triacylglycerol enzymes were affected. To further evaluate the role of PSR1 in regulating lipid and starch metabolism, complementation of psr1 and overexpression by PSR1 was performed. The P-starvation phenotype was clearly rescued in the complementation lines, and overexpression lines showed increased expression of P homeostasis genes and increased Pi accumulation in cells, with an increase in total starch content and number of starch granules. Clear increases in expression of key starch biosynthesis genes such as soluble starch synthase (SSS1, SSS5) and starch phosphorylase (SP1) was observed, which correlated with increased starch content in the overexpression lines. A carbon shift was observed as a decrease in neutral lipid was coupled with the increase in starch content. All together these findings suggest that PSR1 is a key transcriptional regulator of global metabolism, and demonstrated successful transcriptional engineering of microalgae.

Ion exchange resins, commonly used in water treatment, demonstrate promise for the production of biodiesel from biomass feedstocks. The goal of this presented PhD research is to investigate novel uses of ion exchange resins for processing biodiesel feedstocks. Specifically, this research explored using ion exchange resins to remove free fatty acids (FFA) from soybean and waste cooking oils, catalyze transesterification of soybean oil, and catalyze in-situ conversion of dried algal biomass to biodiesel and other recoverable organics. The effect of temperature, moisture content, mixing rate, and resin drying on deacidification of soybean oil with 5% oleic acid feedstock was explored using Dowex Monosphere MR-450 UPW within a batch reactor. The resins were observed to remove up to 83 +/- 1.3% of FFA from soybean oil with less than 5% moisture content while operated at a 20% resin loading at 50 degrees C while mixing at 550 rpm. Once operation characteristics impacting deacidification were evaluated, a series of experiments were carried out to demonstrate the use of mixed bed resin to remove FFA from waste cooking oils. An investigation of wash solutions capable of regenerating the resins was also carried out. Using methanol to regenerate the resins resulted in more than 40% FFA removal over three regeneration cycles, highlighting the utility of resin regeneration as a cost saving measure. Transesterification of soybean oil on Amberlyst A26-OH, a basic ion exchange resin, in the presence of excess methanol was carried out to determine the mechanism of the reaction occurring on the surface. A batch reactor approach was used and reactions were carried out with and without FFA present in the soybean oil feed stock at a 20% resin loading at 50 degrees C while mixing at 550 rpm. When FFA was present in the feedstock and methanol is present in excess, the rate constant for methanol consumption increased. Based upon model fitting, the rate constant of methanol consumption was determined to be 2.08 x 10^-7 /sec with FFA absent and 5.39 x 10^-4/sec when FFA is present when the Eley-Rideal model was used to fit the data. In-situ conversion of dried algal biomass to biodiesel and other recoverable organics was investigated using a batch reaction system with 1 gram of algae. The system was operated with 40:60 methanol:hexane as the solvent system operated at 50 degrees C while mixing at 550 rpm over a range of catalyst loadings. The highest observed ester yield, approximately 60% yield (37 mg_ester/g_algae), was observed when air dried algae was reacted with a 20% resin. An evaluation of the reaction products showed a mixture of esters, phytol, alcohols, and ketones; highlighting the complexity of the reactions occurring during in-situ biomass conversion.

abstract: The continued reliance on fossil fuel for energy resources has proven to be unsustainable, leading to depletion of world reserves and emission of greenhouse gases during their combustion. Therefore, research initiatives to develop potentially carbon-neutral biofuels were given the highest importance. Hydrothermal liquefaction (HTL, a thermochemical conversion process) of microalgae is recognized as a favorable and efficient technique to produce liquid biofuels from wet feedstocks. In this work, three different microalgae (Kirchneriella sp., Galdieria sulphuraria, Micractinium sp.) grown and harvested at Arizona State University were hydrothermally liquefied to optimize their process conditions under different temperatures (200-375 °C), residence times (15-60 min), solids loadings (10-20 wt.%), and process pressures (9-24 MPa). A one-factor-at-a-time approach was employed, and comprehensive experiments were conducted at 10 % solid loadings and a residence time of 30 min. Co-liquefaction of Salicornia bigelovii Torr. (SL), Swine manure (SM) with Cyanidioschyzon merolae (CM) was tested for the presence of synergy. A positive synergistic effect was observed during the co-liquefaction of biomasses, where the experimental yield (32.95 wt.%) of biocrude oil was higher than the expected value (29.23 wt.% ). Co-liquefaction also led to an increase in the energy content of the co-liquefied biocrude oil and a higher energy recovery rate ( 88.55 %). The HTL biocrude was measured for energy content, elemental, and chemical composition using GC-MS. HTL aqueous phase was analyzed for potential co-products by spectrophotometric techniques and is rich in soluble carbohydrates, dissolved ammoniacal nitrogen, and phosphates. HTL biochar was studied for its nutrient content (nitrogen and phosphorous) and viability of its recovery to cultivate algae without any inhibition using the nutrient leaching. HTL biochar was also studied to produce hydrogen via pyrolysis using a membrane reactor at 500 °C, 1 atm, for 24 h to produce 5.93 wt.% gas. The gaseous product contains 45.7 mol % H2, 44.05 ml % CH4, and 10.25 mol % of CO. The versatile applications of HTL biochar were proposed from a detailed physicochemical characterization. The metal impurities in the algae, bio-oil, and biochar were quantified by ICP-OES where algae and biochar contain a large proportion of phosphorous and magnesium. / Dissertation/Thesis / Doctoral Dissertation Chemical Engineering 2020

Microalgae are a diverse group of oxygenic photosynthetic microorganisms which show great promise as a source of biofuel. However, significant challenges still remain before microalgae can be considered a viable source of biofuel. The main current challenges are nutrient sourcing and recycling as well as downstream processing. The algal cell wall and especially the presence of an algaenan cell wall in some Chlorophyte algae could be an important variable in determining downstream processing costs but not much comparative research has been done to elucidate this. The first part of the present study focuses on the recently isolated alga Pseudochoricystis ellipsoidea (Trebouxiophyceae) and its improvement and assessment for biofuel production. Random mutagenesis and FACS screening protocols were developed for the isolation of pigment and cell wall mutants but despite considerable efforts no suitable mutants could be identified in the first half of this project. Two 500 L raceway ponds as well as an algal growth room and bubble column bioreactors were set up to facilitate algal research at the University of Bath and assess the performance of P. ellipsoidea in realistic culture conditions. P. ellipsoidea showed a maximum growth of 1.53 divisions day-1 in semi-open raceway ponds, resistance to contamination and a 30% lipid content, making it particularly suitable for raceway pond cultures. In the second part of this project six species of Chlorophyte (“green”) algae, three of which produced algaenan, were compared for suitability to growth in anaerobic digestate and municipal wastewater as well as cell wall strength, permeability and suitability to hydrothermal liquefaction. We found that anaerobic digestate was a good medium for the growth of all species independently of autoclaving and that non-autoclaved wastewater was a very challenging medium. Algaenan production did not affect cell disruption by ultrasonication but growth stage and cell wall thickness did. Lipid extraction kinetics by chloroform/methanol were greatly affected by algaenan, meaning that this material is relatively impermeable to organic solvents. Cell wall thickness, cell volume and lipid content also had an effect on lipid extraction kinetics but this was only measurable after 180 minutes of extraction. 8 Hydrothermal liquefaction showed high solid and low oil yields, very low sulphur (≤0.1 %) as well as a 1.1 % -1.8 % nitrogen content which is significantly lower than most algal HTL studies to date. This suggests that stationary stage algae are more difficult to process but give a cleaner biocrude and reduce the loss of nitrogen through incorporation in the oil. Significant opportunities for optimisation still exist in the HTL process.