Digital library

Effects of environmental factors such as desiccation, salinity, light and temperature on the diurnal periodicity in liberation of tetraspores in Gelidium pusillum, Pterocladia heteroplatos and Gelidiopsis variabilis were studied. Desiccation of fronds, salinity ard continuous dark or light at different intensities had no effect on the diurnal periodicity in spore output in these three red algae. The temperature of sea wat er was the primary factor controlling the peak output of spores. Peak liberation of spores was delayed for 4-12 hr in a day in G. pusillum and G. variabilis when the temperature of sea water was below 30^oC.

With the exhaustion of fossil-based fuels, microalgae have attracted great interest as a renewable energy feedstock. Microalgae are photosynthetic microorganisms with rapid growth and the potential for production of lipids, proteins, and carbohydrates. However, the capital costs of algae production have been prohibitive for commercial biofuel production. Efforts to further increase algal growth rates and lipid content have attracted significant attention over the past decades to improve biofuel cost-effectiveness. Nevertheless, a fledgling algal industry has emerged in the past decades, but it has primarily focused on protein, nutraceutical, and other high value products from algae. Efforts to improve algal growth rates, however, will benefit nearly all applications of algae. One promising approach is coculturing algae with bacteria to increase algae growth rates and production of biofuel precursors, achieving a win-win outcome. In the research described in this dissertation, efforts were made to improve our understanding of how bacteria alter growth and composition of suspended algae cultures, with a particular focus on plant-growth promoting bacteria (PGPB). 

PGPB, such as Azospirillum brasilense, have the potential to significantly increase algal growth rates through a variety of mechanisms including the production auxin hormones such as indoel-3-acetic acid (IAA). In Chapter 3, a set of lab-scale photobioreactor experiments are described in which the effect of live A. brasilense, exogenous IAA, and spent medium from A. brasilense are studied on two green algae. A. brasilense and IAA were found to promote growth (11-90%) at the expense of energy storage product accumulation in suspended cultures of Chlorella sorokiniana and Auxenochlorella protothecoides. Co-cultures and exogenous IAA stimulated growth in both algae types, but the effect was stronger in C. sorokiniana. These same treatments also suppressed neutral lipids (particularly triacylglycerol) and starch during exponential growth of C. sorokiniana. IAA and co-cultures suppressed starch in A. protothecoides. Spent medium from A. brasilense was also tested and found to promote growth slightly in C. sorokiniana but significant suppress growth in A. protothecoides. It also led to significantly different compositional changes compared to using live A. brasilense, indicating that bioactive constituents in A. brasilense secretions are transient or that physical cell attachment is important for ensuring adequate mass transfer of these constituents. 

The finding that A. brasilense suppressed starch and neutral lipid content of algae raised questions about how A. brasilense mediates oxidative stress in algae. Many algae, including those in this study, are known to accumulate neutral lipid and starch under conditions that induce oxidative stress. Consequently, it was hypothesized that A. brasilense alleviates oxidative stress in algae, thereby promoting growth and suppressing energy storage products. Moreover, PGPB bacteria are known to alleviate the effects of stress conditions in several plants, but the stress- alleviating effects on the algae are not well understood. To evaluate the impacts of A. brasilense on oxidative stress in C. sorokiniana and the consequent changes in biomass composition, algae were co-cultured with A. brasilense under Cu and nitrogen stressors as described in Chapter 4. The results showed that both stressors induced oxidative stress and reduced chlorophyll content. Adding A. brasilense, and to a lesser extent, exogenous IAA, could partially rescue C. sorokiniana from the effects of oxidative stress. In fact, there was no significant difference in ROS levels between nitrogen-limited co-cultures and nitrogen-replete monocultures of C. sorokiniana. This indicates that A. brasilense could rescue the algae from the nitrogen limitation stress, which in turn explained why the presence of A. brasilense led to faster growth, higher chlorophyll content, and lower starch content, as we observed in this study.

The finding that the PGPB, A. brasilense, could promote green algae growth by 11-90%, depending on the algae strain, raised questions about how much more effective PGPB are compared to non-PGPB bacteria. Past research has shown that the non-PGPB, E. coli, can increase algal growth by similar margins. In Chapter 5, a side-by-side comparative study between a PGPB and non-PGPB organism is described. Efforts were made to understand the benefit of “universal” symbiosis mechanisms between algae and bacteria (e.g. cofactor exchange, dissolved O2-CO2 exchange) versus the benefits of PGPB-specific mechanisms (e.g. hormone exchange). The effect of the PGPB, Azospirillum brasilense, the non-PGPB, Escherichia coli, and a recently-isolated strain, Bacillus megaterium, were tested on three green algae: C. sorokiniana UTEX 2714, A. protothecoides UTEX 2341 and C. sorokiniana UTEX 2805. Results showed that, all three bacteria stimulated growth in C. sorokiniana UTEX 2714 and A. protothecoides UTEX 2341, but the effect was stronger in C. sorokiniana. They all led to significantly different compositional changes. Interestingly, the PGPB, A. brasilense slightly suppressed growth in C. sorokiniana UTEX 2805, although the effect was not statistically significant, whereas the other two bacteria significantly increased growth in this strain. This was surprising given that A. brasilense strongly promoted growth in C. sorokiniana UTEX 2714. Additionally, the algae biomass composition, nutrient uptake as well as algal photosynthate changes were measured. The latter indicated significant consumption and cycling of photosynthate, likely generating CO2 for algae. Moreover, the riboflavin metabolite, lumichrome was also detected in co-cultures containing A. brasilense (0.4-0.6 ng/ml) and E. coli (5.5-13 ng/ml). A dose response study showed that lumichrome at 1 to 10 ng/ml led to small but statistically significant increases in growth of C. sorokiniana UTEX 2805 and A. protothecoides. 

Riboflavin metabolites and other vitamin cofactors from a wide range of bacteria likely confer growth benefits to algae. Such mechanisms are present in interactions between algae and both PGPB and non-PGPB. In sum, understanding such coculture relationship details may provide guidance for the cost-effective algae bioenergy and bioproduct development. 

Recent concern over marine pollution has developed great attention on likely alteration of ecosystems thereof. Considering that distributional ranges of seaweed species are governed by their success or failure of reproduction, intertidal green alga, Ulva pertusa, was studied to evaluate reproductive responses to environmental pollutants. Percent of sporulation and spore release of U. pertusa grown in the east seawater medium, optimal photon irrdiance west seawater at irradiances higher than 100μmol·m supper(-2)·s supper(-1). In the east seawater medium, optimal photon irradiance for reproduction was found to be at 100μmol·m supper(-2)·s supper(-1) whereas in the west seawater, that was 30μmol·m supper(-2)·s supper(-1). Total quantum requirements for sporulation and spore release were much lower in the east seawater than the west seawater, which suggests that reproduction may be influenced by water turbidity. When U. pertusa was exposed in batch cultures to various concentrations of nitrate and phosphate at 100μmol·m supper(-2)·s supper(-1) of white light, the rate of reproduction was markedly higher in nutrient-added conditions compared with controls with no nutrients. As nitrate concentration increased, the reproductive rate of U. pertusa increased in all cultures of phosphate concentrations indicating that nitrate but not phosphate plays an important role in reproductive process. When copper and lead in combination were added to U. pertusa, percent sporulation and spore release were solely dependent of copper concentration. Sporulation and spore release at 0.01 ppm of copper reached about 80% which was similar to that in control, but no sign of reproduction was found at 0.1 ppm of copper. It can therefore be speculated that reproduction is more sensitive to copper than growth in U. pertusa in view of the previous report that 0.1 ppm of copper was not inhibitive to growth.

Phenotypic responses of Potamogeton amplifolius and Nuphar advena to different light (7% and 35% of surface irradiance) and nutrient environments were assessed with field manipulation experiments. Higher light and nutrient availability enhanced the growth of P. amplifolius by 154% and 255%, respectively. Additionally, biomass was allocated differently depending on the resource: high light availability resulted in a higher root/shoot ratio, whereas high nutrient availability resulted in a lower root/shoot ratio. Low light availability and high nutrient availability increased the nitrogen content of leaf tissue by 53% and 40% respectively, resulting in a 37% and 31% decrease in the C/N ratio. Root nitrogen content was also increased by low light and high nutrient availability, by 50% (P=0.0807) and 77% respectively, resulting in a 20% and 40% decrease in root C/N ratio. Leaf phenolics were significantly increased 72% by high light and 31% by high nutrient availability, but root phenolic concentrations were not altered significantly. None of these changes in tissue constituents resulted in altered palatability to crayfish. N. advena was killed by the same high nutrient treatment that stimulated growth in P. amplifolius, preventing assessment of phenotypic responses to nutrient availability. However, high light availability increased overall growth by 24%, but this was mainly due to increased growth of the rhizome (increased 100%), resulting in a higher root/shoot ratio. High light tended to increase the production of floating leaves (P=0.09) and significantly decreased the production of submersed leaves. High light availability decreased the nitrogen content by 15% and 25% and increased the phenolic concentration by 88% and 255% in floating and submersed leaves, respectively. These differences in leaf traits did not result in detectable differences in damage by herbivores.