| dc.description.abstract | Microalgae present high potential for greenhouse gases mitigation and value-added biomass production. In this project, the main objective was to characterize genetic and biochemical changes of high CO2 (strain HCA, 50% v/v CO2/air) and low CO2 (strain LCA, atmospheric, 0.04% v/v CO2/air) acclimated strains of Desmodesmus abundans RSM to elucidate adaptation mechanisms to high CO2. Also, a two-stage continuous photobioreactor system for cement flue gas mitigation was designed and optimized; and microalgal biomass was characterized to optimize CO2 capture and propose high-value byproducts of the system. Additionally, strains potential for biological synthesis of silver nanoparticle was evaluated. These objectives were divided into five studies.
In the first study, the genome of D. abundans RSM with an estimated size of 83.61 Mbp was generated. A total of 14 251 genes were predicted, and 58.68 % of these were annotated. Also, carbon fixation pathways were characterized in which ninety-six sequences codifying for twenty-two enzymes were found. Orthologous gene analysis showed 5 414 gene families conserved among D. abundans RSM, Scenedesmus sp. NREL 46B-D3 and species from the Selenastraceae family (M. neglectum and R. subcapitata). Genomic comparison between the strain maintained in our laboratory and the same strain deposited in UTEX Culture Collection of Algae resulted in 902 723 genetic variants which were mainly single nucleotide polymorphisms, and genomes presented 44.36 % of collinear genes. These results evidence genomic changes in microalgae as consequence of adaptation to laboratory conditions; but also conserved gene families between close species in the same taxonomic order (Sphaeropleales).
In the second study, the genome of D. abundans after thirteen years of acclimation to high CO2 (strain HCA) was characterized and compared to the strain LCA. Also, both strains were grown under high CO2 acclimation strategy of six months and, growth and genetic variants were characterized. Assembled genome of strain HCA resulted in an estimated size of 81.20 Mbp, in which 10 535 genes were predicted. Evolution of the strain HCA was characterized by some differentially annotated GO terms to LCA, and in phylogenomic tree that located this strain in a different distance than strain LCA based on 514 single-copy orthologous gene families. Also, it was found only 15.65 % of collinearity between strain genomes and a low number of reads (35.70%) from strain HCA that mapped in LCA genome. Evaluation of high CO2 acclimation strategy for six months showed that strain LCA reached similar growth than strain HCA by the end of the experimental period. Microalgae adaptation to high CO2 involved higher accumulation of genetic variants (130 082) than under air (107 163), resulting in estimated mutation rates of 3.17x10-5 and 2.85x10-5 per base per generation, respectively. Strain HCA under air presented higher difficulties to adapt to this condition and accumulated 119 334 variants in air and 82 360 in CO2 with mutation rates of 3.56x10-5 and 2.25x10-5 per base per generation. High CO2 acclimation strategy of six months represents a simple and rapid way to generate higher CO2 tolerant strains.
In the third study, strains LCA and HCA were grown in different N-concentrations under a continuous flow of high CO2. Growth, biomass composition and gene expression of N-transporters and biosynthesis of starch and triacylglycerol were characterized. Maximum cell concentration increased for strain HCA at higher N, but not for LCA. Also, only strain LCA showed an adaptation phase of 24 h for all conditions, and higher N intake rates were determined in HCA. Biomass productivities were not significant different among N concentrations and strains, ranging 0.097-0.134 g d.w. L-1 d-1. Similar biomass productivities evidence microalgae carbon allocation into different metabolites and therefore similar CO2 capture efficiencies. In both strains, under the lowest N (6 mg L-1) starch accumulated up to 12.7-14.52 % d.w., which in high N (50-250 mg L-1) was 5.1-7.1 % d.w. Contrary, protein decreased as N decreased in the medium from 43.3-54.8 % d.w to around 14 % d.w. While accumulation of neutral lipids occurred only in strain LCA for all N conditions. In conclusion, after thirteen years under low and high CO2 acclimation, strains present differential response to N concentration under high CO2. Growth and composition in strain LCA were affected by N and high CO2, while HCA only by N availability. This was also evident in gene regulation, where HCA presented a faster response to growth condition than LCA.
A fourth study with strain HCA, evaluated the growth and nutrient utilization (CO2, NOx and SOx) of this strain under a model cement flue gas (MFG; 25% CO2, 700 ppm NO, and 100 ppm SO2) using a limited N and without S medium (BG11-0.2N-S) and complete medium (BG11) in an optimized two-stage continuous photobioreactor system (TSCB). Also, biomass composition (starch, protein, pigments and lipids) and productivities; as well as fatty acid profile were characterized. Microalga tolerated and used flue gas as nutrient source when using the TSCB system, and pH was successfully controlled through system stages by adding 150 mg L-1 d-1 of cement kiln dust (CKD). Biomass productivities was around 1.2 g L-1 d-1 for both culture medium. When using BG11-0.2N-S medium 26% lower protein than in complete BG11 were obtained, with productivities of 0.46 ± 0.05 and 0.63 ± 0.05 g L-1 d-1, respectively. Contrary, starch and lipid were higher in low N medium. Pigment concentrations were 1.3 to1.4-fold higher in complete medium. Fatty acid profile showed higher content of PUFA in free fatty acid fraction under limited N, and in di and mono- glycerides under repleted N condition. Biomass and metabolite productivities were improved by using the TSCB system compared to batch cultivation strategies. Target metabolite accumulation was induced by using different N concentrations, where limited N in the second stage allocate carbon into starch and lipids, while medium with extra N into protein and pigments.
In the last study, the potential of D. abundans strain LCA and HCA was evaluated as a suitable platform for silver nanoparticle (AgNPs) synthesis. The effect of biological components, namely, cell pellet, supernatant, and both components, were compared to the culture collection strain Spirulina platensis at different pH values. All biological components of strain HCA at pH 11 showed potential for nanoparticle synthesis. AgNPs (14.9 ± 6.4 nm diameter) with the lowest charge (-32.7 ± 5.3 mV) were observed using the cell pellet and, preserving the supernatant, resulted in synthesis of AgNPs in all pH solutions. In contrast, no nanoparticles were observed with components of strain LCA, except for the cell pellet at pH 11 (127.8 ± 14.8 nm, -26.7 ± 2.4 mV). The reducing power in strain HCA might be attributed to functional groups from proteins, carbohydrates, and fatty acids; and, in the supernatant, to amino acids, monosaccharides, disaccharides, and polysaccharides. Finally, AgNPs of the three microalgae strains exhibited similar antimicrobial properties against E. coli in the agar diffusion test. It is suggested that the high CO2 atmosphere potentiates biological components in D. abundans strain HCA, which might benefit their use in nanotechnology and represent an exciting byproduct from CO2 mitigation systems. | |
