Algae-associated bacteria in photobioreactors

Research described in this thesis aimed to investigate the microalgal microbial ecology by integrating both cultivation-dependent and cultivation-independent approaches. In addition, we examined effects of bacteria on growth of microalgae by developing a coculture system. Understanding the bacterial community composition and functional associations between microalgae and bacteria will provide important insights for both aquatic ecology and algal biotechnology.

Microalgae are living with diverse microbial communities and interactions between microalgae and bacteria are prevalent and strongly influence carbon and nutrient cycling in aquatic ecosystems. A brief overview of the current knowledge of algae-bacteria interactions and current research methodologies was given in chapter 1. This chapter furthermore introduced the microalgae production facilities at AlgaePARC, and highlighted two research objects (Nannochloropsis sp. and Botryococcus braunii), and current knowledge on their associated bacteria.

In chapter 2 we comprehensively reviewed the recent research progress on algae-bacteria interactions and summarized the current knowledge on functional aspects of algae-associated microbiomes. In addition, we discussed the applications of knowledge on algae-bacteria interactions in algal biomass production and various other related biotechnological innovations. Finally, we concluded that principles of algae-bacteria interactions can be integrated into many aspects of the algal production chain, which may help push the limit of the algal industry.

In chapter 3, we studied the bacterial community associated with 12 strains of B. braunii. The changes of bacterial community composition were monitored over a period of 12 days. It was clear from this study that B. braunii hosts a wide variety of bacterial species, among which the bacterial families Rhizobiaceae, Bradyrhizobiaceae and Comamonadaceae were found in all 12 strains. These families that all belong to the phylum Proteobacteria could have important interactions with B. braunii. Additionally, although each strain displayed a different bacterial community composition, all the strains from the CAEN culture collection had more similar bacterial communities, suggesting that the algae culture collection could have an influence on the bacterial community composition. Several bacterial genera were shown to be exclusively abundant in CAEN strains including Rhizobium spp. and Porphyrobacter spp.

In chapter 4, we compared bacterial community composition in cultures of Nannochloropsis sp. CCAP211/78 grown simultaneously in four outdoor large-scale bioreactors. We observed that the bacterial community composition significantly differed between all reactor types except for horizontal and vertical tubular bioreactors. Although the bacterial communities  varied within each bioreactor at different cultivation stages, we found that a member of the family Saprospiraceae was the most abundant taxon (OUT_1261) in all large non-sterilized bioreactors. This indicates that Saprospiraceae may play important roles in Nannochloropsis cultures.

In chapter 5, we isolated bacteria from one horizontal tubular bioreactor and one vertical tubular bioreactor. Among all the isolates eighteen bacteria were phylogenetically classified as different species/strains based on their 16S rRNA gene sequence. Axenic cultures of Nannochloropsis sp. CCAP211/78 were successfully obtained by treatment with antibiotics. A microplate-based assay system coupled with a custom-made LED box was developed to assess the growth of axenic Nannochloropsis sp. CCAP211/78 with addition of isolated bacterial strains. Out of 18 strains, one Maritalea porphyrae strain (DMSP31) and one Labrenzia aggregata strain (YP26) were highlighted as having a significantly positive effect on microalga growth, whereas one flavobacterial strain (YP206) was shown to reduce this growth. YP26 was also found to substantially enhance the growth of Nannochloropsis sp CCAP211/78 using the double-agar plate assay, which indicates that YP26 has strong positive impacts on Nannochloropsis sp CCAP211/78.

Finally, chapter 6 elaborated on the implications of findings from the research described in this thesis. Bacterial community composition of Nannochloropsis sp CCAP211/7 and B. braunii was compared, and the results generally supported that bacterial communities are algal species specific. Additionally, I suggested that the combination of environmental variables and reactor configuration/operation (in addition to stochastic effects) may be responsible for the differences in bacterial community composition observed between different reactor types and between runs. Further exploration and exploitation of algae-bacteria interactions is needed. Therefore, I proposed several new techniques, including using a new primer set to better characterize bacteria living with microalgae, using advanced mass spectrometry to tap into the metabolic changes of microalgae when they interact with bacteria, and using microfluidics coupled with Raman-based cell sorting to isolate bacteria of interest. Future research should consider these approaches in combination with coculture experiments in order to gain better understanding on bacterial community composition and functional interactions between microalgae and bacteria.