1. Algae Culture 2011-20121 Chapter 7: Interactions between microalgae and bacteria.

2. Algae Culture 2011-20122 Rhizosphere Phycosphere Microenvironment containing both Prokaryotes (Eubacteria, Archaea) and Eukaryotes (Protozoa, Fungi, Algae, Metazoa). Interactions between all organisms are crucial.

3. Rhizosphere

4. Algae Culture 2011-20124

5. 5 Uni-algal, non-axenic cultures of six marine diatoms were screened by polymerase chain reaction/denaturing gradient gel electrophoresis for the diversity of the accompanying bacterial communities (‘satellite’ bacteria) in order to test the hypothesis that algal cells constitute niches for specific bacterial species. Analysis of replicate incubations and repeated passage of cultures in most cases showed only minor variations in satellite assemblage genetic fingerprints, suggesting that the bacterial/algal associations were stable.

6. Algae Culture 2011-20126

7. 7 Most of the populations represented typical marine phylotypes, such as members of the α-Proteobacteria (related to the genera Ruegeria, Sulfitobacter, Roseobacter and Erythrobacter), or members of different genera of the Cytophaga-Flavobacterium-Bacteroides (CFB) phylum. Surprisingly, β-Proteobacteria were also found in two of the cultures. A common point for all cultures was the presence of at least one representative of the α-Proteobacteria and of the CFB phylum, both of which have been reported as important representatives of the marine picoplankton. Their ubiquity in the sea and in the phytoplankton cultures analysed points to a specific role of these bacteria in the marine food web. The results indicate that algal diversity might be an important factor in explaining the enormous bacterial diversity in marine assemblages, and vice versa. Specific substances in the photosynthetic extracellular release and in the organic carbon produced by different phytoplankton species may require a variety of bacterial populations for the processing of this algal-derived organic matter.

8. Algae Culture 2011-20128

9. 9 Heterotrophs cannot synthesize critical nutrients and thus must depend on certain extrinsic nutrients from photoautotrophs. Photosynthetic metabolites from photoautotrophs play an important role in their symbiotic association with heterotrophs in the environment. extracellular-released organic carbon (EOC) produced by Chlorella sorokiniana IAM C-212 could play an important role as a nutrient source for symbionts (CSSB- 1, CSSB-2, CSSB-3 and CSSF-1) separated from unialgal rather than axenic strains of C. sorokiniana IAM C-212. Using partial 16S or 18S rRNA gene analysis, CSSB-1, CSSB-2, CSSB-3 and CSSF-1 were shown to be highly similar to various microbial strains: Ralstonia pickettii (AB004790) (99.8% identity), Sphingomonas sp. DD38 (AF105023) (99.4% identity), Microbacterium trichotecenolyticum [Y17240] (98.6% identity), and Acremonium-like fungus [AB108787] (98.8% identity). The association between these symbionts and C. sorokiniana was facultative rather than obligate symbiosis.

10. Algae Culture 2011-201210 Algal extracellular products are composed of carbohydrates, nitrogenous compounds, organic acids, lipids and vitamins, among other components. However, a detailed knowledge of the chemical composition of photosynthetic metabolites released by an algal strain and utilized by heterotrophs (symbionts) is lacking. In this study, we performed qualitative and quantitative analyses of components of the EOC excreted by C. sorokiniana IAM C-212, and studied the consumption of these components by algal symbiotic heterotrophs. In addition, a novel artificial EOC (A-EOC) medium was developed to imitate the nutritional conditions of the natural environment surrounding plant or algae. The composition of this medium was based on analysis of the relationship between the green algae Chlorella and its symbionts in relation to photosynthetic metabolites. Based on these developments, we propose a new approach for developing cultivation media.

11. EOC:quantification After cultivation, the TOC concentration, which is representative of the EOC concentration, was 307 mg C per liter and the algal cell concentration in the cultivation broth was 11 g dry wt. cells per l or roughly 3% The concentration of free dissolved carbohydrate which could be analysed qualitatively and quantitatively without hydrolysis was 23 mg C per liter, about 7% of the TOC concentration. The concentration of the detectable free dissolved organic nitrogen was 10 mg C per liter, about 3% of the TOC concentration.

12. Algae Culture 2011-201212 Chemical standards of the various carbohydrates used for analysis included xylitol, myo-inositol, l-fucose, l-rhamnose, d-arabinose, d-galactosamine, d- glucosamine, d-galactose, d-glucose, d-xylose, d-mannose, d-fructose, d-ribose, melibiose, sucrose, d-galacturonic acid and d-glucuronic acid. Free dissolved carbohydrates were separated by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD).

13. Algae Culture 2011-201213

14. Algae Culture 2011-201214 Analysis of the free dissolved organic nitrogen was performed using a JLC- 300 amino acid analyzer. Amino acid standard solutions were used as chemical standards.

15. Algae Culture 2011-201215

16. Algae Culture 2011-201216 Consumption of free dissolved carbohydrates and organic nitrogen in EOC by algal symbionts: Half-strength freshly prepared MM4N photoautotrophic medium (inorganic basal medium) was filtered through a sterile 0.22µm pore size filter unit and then mixed with an equal volume of EOC prepared from algal culture broth. This mixture was considered as ‘natural’ EOC medium (N-EOC medium). Each symbiotic microorganism (CSSB-1, CSSB-2, CSSB-3 and CSSF-1) isolated previously was inoculated into 5 ml of N-EOC medium in a test tube. Control cultures were not inoculated with the symbiotic micro-organisms. After inoculation, each culture was incubated at 30°C for 3 days and each culture was centrifuged (5000 g, 15 min at 4°C) to remove the cells and filtered through a sterile 0.22µm pore size filter unit. Consumption of free dissolved carbohydrate and organic nitrogen in the N-EOC medium was analysed with a HPAEC-PAD and an amino acid analyser.

17. Algae Culture 2011-201217

18. Algae Culture 2011-201218

19. Algae Culture 2011-201219