Algae-bacteria interactions during commercial production of Nannochloropsis sp. in closed photobioreactors Javier Giraldo Academic Year 2018-2019 Promoters: Prof. Dr. Wim Vyverman Dr. Luc Roef Dissertation submitted in fulfilment of the requirements for the degree of Doctor (Ph.D.) in Sciences: Marine Sciences (Ghent University) 1 2 Members of the examination board Voting members: Prof. Dr. Koen Sabbe, Ghent University, Belgium (Chair) Prof. Dr. Anne Willems, Ghent University, Belgium (Secretary) Prof. Dr. Ir. Sven Mangelinckx, Ghent University, Belgium Prof Dr. Koenraad Muylaert, KU Leuven-Kulak, Belgium Prof. Dr. Peter Kroth, Konstanz University, Germany Dr. Peter Chaerle, Ghent University, Belgium Non-voting members: Dr. Luc Roef, Proviron Holding NV Prof. Dr. Wim Vyverman, Ghent University This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No 642575. This work makes use of resources and facilities provided by UGent as part of the Belgian contribution to EMBRC-ERIC. 3 4 Contents Summary....................................................................................................................................................................................... 9 Samenvatting ........................................................................................................................................................................... 13 Chapter I .................................................................................................................................................................................... 17 General introduction and thesis objectives ............................................................................................................ 17 1. General introduction to industrial algal cultivation ............................................................................. 18 1.1. Potential algae applications ....................................................................................................................... 18 1.2. Cultivation of microalgae ............................................................................................................................ 19 2. Algae production for aquaculture at Proviron ....................................................................................... 22 2.1. Characteristics of Nannochloropsis sp. .................................................................................................. 22 2.2. The ProviAPT system ................................................................................................................................... 23 2.3. Exploiting algae potential and overcoming limitations ................................................................. 24 2.3.1. Reactor design ............................................................................................................................................ 25 2.3.2. Operating conditions ............................................................................................................................... 25 2.3.3. Dealing with biofouling at Proviron .................................................................................................. 26 3. The algal microbiome: algae-bacteria interactions .............................................................................. 28 4. Objectives and outline of this thesis ........................................................................................................... 29 Chapter II ................................................................................................................................................................................... 33 Exploration of the algal microbiome during industrial cultivation of Nannochloropsis sp. in closed photobioreactors and its potential effect on lipids composition .................................................................. 33 Abstract ............................................................................................................................................................................ 34 1. Introduction .......................................................................................................................................................... 35 2. Materials and methods ..................................................................................................................................... 35 2.1. Algae cultivation and sample selection ................................................................................................. 35 2.2. Sample selection and characterization ................................................................................................. 36 2.3. Fatty acids profiling ...................................................................................................................................... 37 2.4. Microbial characterization ......................................................................................................................... 38 2.5. Statistical analysis ......................................................................................................................................... 38 3. Results and discussion...................................................................................................................................... 39 3.1. Reactor characterization and sample selection ................................................................................ 39 3.2. Total fatty acids content and fatty acids profile ................................................................................ 42 3.3. Microbial diversity ........................................................................................................................................ 44 3.4. Correlations between microbial diversity and EPA content ........................................................ 49 4. Acknowledgements ............................................................................................................................................ 51 5 5. Supplementary material .................................................................................................................................. 52 Chapter III ................................................................................................................................................................................. 61 Influence of the algal microbiome on biofouling during industrial cultivation of Nannochloropsis sp. in closed photobioreactors ..................................................................................................................................... 61 Abstract ............................................................................................................................................................................ 62 Keywords ......................................................................................................................................................................... 62 Highlights ......................................................................................................................................................................... 62 1. Introduction .......................................................................................................................................................... 63 2. Materials and methods ..................................................................................................................................... 65 2.1. Biofilm sampling protocol and selected samples ............................................................................. 65 2.2. Microbial isolation ......................................................................................................................................... 66 2.3. Bacterial identification by MALDI-TOF MS and 16S rRNA gene sequencing ........................ 67 2.4. Isolation, treatment and identification of an invasive green algae species ........................... 67 2.5. Algae cultivation in shake flasks .............................................................................................................. 68 2.6. Co-cultivation of Nannochloropsis sp. with the green alga species ........................................... 68 2.7. Biofilm formation assay .............................................................................................................................. 68 2.8. Statistical analysis ......................................................................................................................................... 69 3. Results and discussion...................................................................................................................................... 70 3.1. Microbial isolation ......................................................................................................................................... 70 3.2. Effect of the microbiome on the productivity of Nannochloropsis sp. ..................................... 75 3.3. Poseidonocella sp. is the strongest biofilm inducer among 24 isolated bacterial strains 78 4. Conclusions ........................................................................................................................................................... 79 5. Acknowledgements ...........................................................................................................................................