## 4 Bacteria Associated with _Haslea ostrearia_  
In a benthic biofilm microenvironment, bacteria and microalgae have close mutualistic, commensal or parasitic interactions [76]. They connect with each other, both having major contributions to the different biogeochemical cycles in the ocean [77; 78; 79]. These auto- and heterotrophic organisms rely on each other for cross feeding [80], and the molecules mainly exchanged are sources of carbon, nitrogen, vitamins (cobalamin), iron and other trace elements. In natural environments, diatom communities vary depending on abiotic factors--such as temperature, salinity, nutrients, etc. [81; 82; 83]--and biotic factors, such as competition between diatoms (allelopathy) [37] and the associated bacteria [84]. However, bacteria can also influence diatom communities by interfering with nutrient availability and their reproduction cycle [85; 86]. In the case of clonal cultures, the composition of the bacterial community varies according to the strain/species and the physiological state of the culture [87]. The variation of the bacterial community can also be linked to the excretion of exopolymeric substances (EPSs) by diatoms, as EPSs depend on species and environmental conditions [87; 88; 89]. In our study, we aimed to produce a clonal culture of _H. ostrearia_ strain HoB4 to facilitate the completeness and accuracy of the genomic assembly. To this end, we applied various antibacterial and antimyotic agents (see the section on DNA isolation above). Even after these preventive measures, most of the DNA in the samples was of bacterial origin (>70% of Illumina reads). Eight different circular sequences were detected with the 16S region V1-V3, with two of them corresponding to HoB4 mitochondrial and chloroplast genomes. The bacterial sequences were more abundant than the mitochondrial and  
Figure 3: Flowchart of the bioinformatic pipeline used to assemble the HoB4 genome. chloroplast ones, at 79%, 3% and 18%, respectively. The majority of the bacteria detected belong to the branch of _Proteobacteria_ (57%), followed by the branch of _Bacteroidetes_ (24%) and _Cyanobacteria_ (18%) (Figure 4). Complete genomic sequences were found for the genera _Maribacter_ (Flavobacteriumaceae), _Mycobacterium_ (Mycobacteriaceae), _Paracoccus_ (Rhodobacteraceae), _Pseudomonas_ (Pseudomonasaceae), and _Sphingorhabditis_ (Sphingomonadaceae). Interestingly, genera belonging to Flavobacteriaceae, Pseudomonas, Pseudomonas, Pseudomonas, Pseudomonas, and Sphingomonadaceae have already been found to be associated with diatoms [94; 95]. This also concerns the genus _Ilumatobacter_ (Ilumatobacteraceae) and some cyanobacterial genera, for which we have detected only partial sequences. Furthermore, these taxa are found in sea- or fresh-water, and are commonly found in diatoms' microbiomes. For the above reasons, these bacteria seem to be tightly associated with _H. ostrearia_ strain HoB4, which would explain why they are difficult to remove without killing the diatom partner. Nevertheless, there could be one exception regarding the presence of _Pseudomonas_, as this genus of Gammaproteobacteria is usually found in waste water [96]. The presence of this genus in the sample could result from contamination prior to sample collection. However, it could also be a true satellite of the HoB4 microbiome under natural conditions. Irrespective of their origin, the presence of bacterial reads/sequences in the sample hampered the assembly of the genome of _H. ostrearia_ strain HoB4. Without clean, axenic data and/or previous knowledge about the studied species, it is difficult to identify which sequence belongs to which organism. Their abundance even causes some troubles when  
Figure 4: Krona plot of the microbiome detected by searching 16S amplicons in HoB4 metagenomic assembly. The plot displays relative abundance and hierarchy with the use of a radial space-filling display. The amplicons were searched with primers targeting the V1-V3 region of the 16S rRNA gene [90].