The economics of microalgal bioproduct commercialization would benefit from increased accumulation of energy storage compounds, such as carbohydrates and lipids, and increased biomass productivity. This thesis explores two adaptive laboratory evolution strategies for improving Scenedesmus obliquus: single UV-mutagenesis and low light cultivation at a high dilution rate to produce cultigens with greater carbohydrate content and iterative UV-mutagenesis and selection under outdoor conditions in a raceway pond at a high dilution rate to increase biomass productivity. Two cultigens were generated with the goal of increased carbohydrate content: K5 and K7. Both were mutagenized for 5 seconds and cultivated in 50-mL Klavins reactors. K5 was maintained at 95 μmol/m2-sec for 16 hours/day, and K7 was maintained at 250 μmol/m2-sec for 6 hours/day. When evaluating the two cultigens in triplicate 800-mL bubble columns against wild-type Scenedesmus obliquus, neither K5 nor K7 demonstrated an increase in carbohydrate content. To increase biomass productivity, Scenedesmus obliquus was iteratively UV-mutagenized (aliquots exposed for 5-60 seconds were combined) and cultivated in a 100-L, 0.5-m2 outdoor raceway pond fed with filtered reclaimed wastewater at a high dilution rate. Three rounds of mutagenesis and selection took place during the spring, summer, and winter months, with the MBE 509 and MBE 510 cultigens being produced after the summer and winter selection rounds, respectively. When evaluated in triplicate 800-mL bubble column reactors: MBE 509 (491 ± 42 mg/L-day, expressed as the mean of the time series ± SD) was 24% more productive than wild-type (397 ± 39 mg/L-day), and MBE 510 (443 ± 26 mg/L-day) was 13% more productive than wild-type (391 ± 9 mg/L-day). When evaluated in replicate 1,350-L, 4.5-m2 raceway ponds, MBE 509 (15.2 ± 2.2 g/m2-day) and MBE 510 (16.1 ± 1.8 g/m2-day) were 5% and 12% more productive than wild-type (14.5 ± 2.4 g/m2-day), respectively. Selection under low light and high dilution did not result in cultigens with higher carbohydrate content relative to wild-type Scenedesmus obliquus, but iterative mutagenesis and selection in an outdoor raceway pond did generate cultigens with higher biomass productivity in both indoor and outdoor environments.

The economics of algae biofuels and bioproducts would be improved by increased biomass productivity. Two studies on this potential are described in this thesis – one on a locally isolated filamentous yellow-green alga and the other on a planktonic strain genetically improved via selective enrichment. Polycultures have been viewed as productive, stable, and, in some cases, harvestable by natural bioflocculation. Local native strains might have higher productivity than culture collection strains because they are already adapted to local outdoor conditions. In this study, the filamentous yellow-green alga Tribonema minus was isolated from a local volunteer polyculture. Its productivity as a monoculture was compared to a volunteer polyculture in a year of thrice-weekly samples. The study was conducted in duplicate 1,000-L, 3.5-m2 outdoor raceway ponds fed with nitrified and filtered reclaimed wastewater. T. minus monocultures were more productive (17.6 ± 0.5 g/m2-d; mean ± range) than the polyculture (13.3 ± 0.4 g/m2-d). The T. minus monocultures were stable, growing for an average of 38 days before significant contamination with other algae genera, at which point the cultures were restarted. The annual average biochemical composition, in percent of ash-free dry-weight, of the T. minus cultures was 28.3 ± 0.4% (mean ± std. dev.) carbohydrates, 37.6 ± 0.7% proteins, and 6.1 ± 0.3% lipids. Eicosapentaenoic acid, a valuable nutritional omega-3 fatty acid, comprised 0.3% to 4% of the ash-free dry-weight and was the predominant fatty acid methyl ester measured. In summary, an alga isolated from a volunteer polyculture was more productive as a monoculture than the originating polyculture. The monoculture biomass contained a valuable nutritional fatty acid. Scenedesmus obliquus was subjected to UV mutagenesis followed by cultivation in benchtop bubble columns at high dilution rates to select for cultures (cultigens) that grew faster than the wild-type. Fast growing cultigens were transferred to 1,350-L outdoor raceways ponds for productivity measurement. Cultigen and wild-type cultivation was conducted on reclaimed wastewater media in coastal central California for seven months. One cultigen, MBE 501, had 23% higher productivity than the S. obliquus wild-type (11.5 ± 0.02 vs. 9.4 ± 0.6 g/m2-d) during July 28-December 30, 2019. MBE 501 had been subjected to 1:400 and 1:200 dilutions twice per week for the first two months and last five months of selection, respectively, and went through 289 generations in the lab. Compared to a volunteer polyculture (14.4 ± 1.3 g/m2-d), MBE 501 was not as productive on average. This study demonstrated that high dilution rates in lab cultures can select for cells that are more productive in outdoor raceways. Genetic comparison of MBE 501 and its wild-type are pending.