Botryococcus braunii for the production of hydrocarbons and exopolysaccharides and the role of associated bacteria

Microalgae are photosynthetic organisms that are found worldwide in many different aquatic environments and therefore display an immense biological diversity. They are a promising source of many useful polymers that have industrial applications such as food, fuel, material and pharmaceutical. One microalga that has gathered quite a research community is Botryococcus braunii. The reason for its scientific club is the fact it can synthetize long chain hydrocarbons molecules from C20 to C40. These hydrocarbons have been found in oil-shales and tests show that it can be used as aviation fuel. Besides producing hydrocarbons, some strains of B. braunii can produce exopolysaccharides (EPS) composed mainly of galactose and a small fraction of fucose. The EPS has interesting rheological properties for the food industry and potential active compounds that could be used in the pharmaceutical industry . Like many other microorganisms, microalgae in the natural environment are usually in the presence of bacteria. The presence of bacteria with microalgae can either have a beneficial or an antagonistic effect. For B. braunii little is known about the bacteria community present especially for the EPS producing strain. For that reason, the aim of this thesis was to investigate B. braunii’s associated bacteria with the aim of improving B. braunii’s biomass growth and hydrocarbon and EPS content. In chapter one, we introduced the topic of microalgae as a potential source of sustainable polymers and we introduced the species B. braunii, describing its characteristics and scientific interest. It is also introduced the topic of microalgae and bacteria associations by looking at other studies from literature. In chapter two, 16 publically available strains of B. braunii were ordered in culture banks and screened for biomass productivity, hydrocarbon and total carbohydrate content. The aim of the study was to identify one or more good strains that displayed high biomass productivity as well as hydrocarbon or total carbohydrate content. In seven strains out of 16 cultivated in 250 mL volume Erlenmeyer flaks, we detected 5 to 42 % content of hydrocarbons of the dry biomass with four strains producing botryococcenes (C30-C34) and three strains producing alkanes (C20-C25). Two strains showed high amounts of EPS content above 50 % per dry biomass. Seven strains comprising of the strains with higher biomass productivity plus the highest hydrocarbons and EPS content, were tested for scalability using bench scale 800 mL volume bubble column reactors. Two strains, AC761 which produces botryococcenes and CCALA778 which produces EPS, were selected as the most promising B. braunii strains for industrial production of hydrocarbon and EPS. In chapter three, we studied the bacterial community associated with B. braunii. We cultured 12 strains from the initial 16 from chapter 2 and extracted the DNA from samples taken over a time period of 12 days. It was clear from this study that B. braunii hosts a variety of bacterial species and still maintain its growth. The bacteria families Rhizobiaceae, Bradyrhizobiaceae and Comamonadaceae were found in all 12 strains. These families which belong to the phylum Proteobacteria could have an important role regarding B. braunii growth. Each strain displayed a different bacterial community composition but all the strains from the CAEN culture collection clustered near each other suggesting that the algae culture collection could have an influence on the bacterial community composition. Bacteria genus identification based on 16S rRNA gene amplicon similarity showed several genera present including Rhizobium spp. and Variovorax spp.. Two genera were found that are possibly linked to hydrocarbon degradation: Sphingomonas spp. and Rhodobacter spp.. In chapter four, we investigated further B. braunii CCALA778 which was shown in chapter 2 to accumulate high amounts of EPS. We investigated the effects of antibiotics on algal growth, EPS accumulation and bacterial community composition of CCALA778. Taxonomical identification by 16S rRNA gene analysis indicated that most of the bacteria present with CCALA778 were Gram-negative. Of all antibiotics and antibiotic mixes, only the treatment with Penicillin did not affect the growth of B. braunii. The remaining antibiotics halted the growth of CCALA778 while they were active. The exceptions were with the antibiotics Chloramphenicol, Gentamycin and Linezolid which permanently ceased the growth of CCALA778. The accumulation of EPS seemed to be related to biomass growth, but we did also observe a reduction of EPS with the cultures treated with Penicillin suggesting that bacteria could have an effect on the EPS content. Antibiotics had specific effects on the bacterial community with all treatments showing significant changes over time. The most efficient treatment in removing bacteria were the mixes Metronidazole-Rifampicin-Penicillin and Penicillin-Rifampicin which were the only treatments to show significant changes in the bacterial community when compared to the untreated cultures after 10 days of cultivation. Antibiotics and antibiotic mixes can create changes in the bacterial community but it is unlikely that they alone can lead to axenic B. braunii cultures. In chapter five, we used Ultra Violet-C light (UVC) to reduce bacteria diversity and abundance present in B. braunii CCALA778. UVC is highly effective in inactivating bacteria and for that reason is being investigated further in medicinal applications. After applying the UVC to B. braunii CCALA778, we were able to reduce the relative abundance of 16S rRNA genes assigned to bacteria to less than 1 % compared to the 70% in the non-treated cultures. With the UVC treated CCALA778 we observed several physiological changes. The UV treated cultures with reduced bacterial load showed nearly double the EPS accumulation when compared to the untreated. To confirm that we did not see an artefact in our results due to the UVC treatment, UVC treated cultures were also inoculated with bacteria from the untreated and we observed a reduction of EPS similar to what we saw with the untreated cultures. There were no changes to the EPS composition after the removal of the bacteria. Other physiological changes were observed, namely that colony size of B. braunii CCALA778 significantly increased when compared to the untreated culture and the UV treated with bacteria. We hypothesise that the increase in colony size was probably due to the fact there was more EPS accumulated which helped with cell aggregation. We also observed an increase on the biomass growth in the UV-treated CCALA778 which we hypothesized being related to the fact that there was none or hardly any competition for essential micronutrients such as phosphate. From this study we concluded that the associated bacteria present with B. braunii CCALA778 were antagonistic. We believe the reason why the bacteria were antagonistic is because of the readily available EPS which is a rich source of organic compounds that bacteria could use for their own proliferation allowing them to compete with B. braunii for essential nutrients. In chapter 6, we discuss the implications from our previous 4 experimental chapters. The aim of the study was to improve the biomass productivity and hydrocarbon and EPS content of the microalgae B. braunii. In brief, B. braunii displayed a wide range of physiological traits regarding biomass productivity and hydrocarbon and total carbohydrate content. We showed that B. braunii can co-habit with a wide range of bacteria diversity and abundance and that the associated bacteria were antagonistic to CCALA778 by affecting its biomass growth. We also showed that by removing the associated bacteria we can increase the EPS accumulation. Currently most of the research on microalgae and bacteria interactions, focus on the positive side, but we must understand also how bacteria can be antagonistic to microalga growth. Bacteria can be antagonistic to microalgae by competing for nutrients and also being detrimental to industrial process by degrading the product of interest in the case of organic carbons such as EPS. Therefore it is unlikely we can use the benefits that bacteria can provide such as enhancing growth to improve the cultivation of B. braunii and other similar microalgae species that secrete EPS. Since bacteria can be antagonistic to microalgae that secrete large amounts of organic compounds such as EPS, it is imperative to minimize contamination in large scale photobioreactors (PBR). It is important because in large scale PBR, contamination can occur leading to downtime of the reactors. If microalgae industry is to advance, it must develop PBR units that prevent contamination of bacteria from the surrounding environment.