Effects of growth-promoting bacteria in suspended green algae cultures for bioenergy and bioproduct development

Abstract

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.