Phytoplankton consortia as a blueprint for mutually beneficial eukaryote-bacteria ecosystems based on the biocoenosis of Botryococcus consortia

Bacteria occupy all major ecosystems and maintain an intensive relationship to the eukaryotes, developing together into complex biomes (i.e., phycosphere and rhizosphere). Interactions between eukaryotes and bacteria range from cooperative to competitive, with the associated microorganisms affecting their host`s development, growth and health. Since the advent of non-culture dependent analytical techniques such as metagenome sequencing, consortia have been described at the phylogenetic level but rarely functionally. Multifaceted analysis of the microbial consortium of the ancient phytoplankton Botryococcus as an attractive model food web revealed that its all abundant bacterial members belong to a niche of biotin auxotrophs, essentially depending on the microalga. In addition, hydrocarbonoclastic bacteria without vitamin auxotrophies seem adversely to affect the algal cell morphology. Synthetic rearrangement of a minimal community consisting of an alga, a mutualistic and a parasitic bacteria underpins the model of a eukaryote that maintains its own mutualistic microbial community to control its surrounding biosphere. This model of coexistence, potentially useful for defense against invaders by a eukaryotic host could represent ecologically relevant interactions that cross species boundaries. Metabolic and system reconstruction is an opportunity to unravel the relationships within the consortia and provide a blueprint for the construction of mutually beneficial synthetic ecosystems.


Biocoenosis of Botryococcus consortia
. Metagenome datasets were taxonomically assigned via MEGAN 1 (200,000 randomly subsampled reads, minimum 50 reads per taxonomic group). The relatively high proportion of unassigned metagenomic reads likely results from the fact that the algal and some bacterial genomes are not sequenced and disposed in the used NCBI-NR 2 database. The analysis revealed the occurrence of the phyla Euryarchaeota and Viruses belonging to the family of Microviridae (bacteriophages with a singlestranded DNA genome). Illustration of the relative abundance and phylogenetic distribution of bacterial genera detected via high-throughput 16S rDNA amplicon sequencing approach (for details see Table S3).  (Table S4) per analyzed genome (bold).

Biocoenosis of Botryococcus consortia
Black, grey and white circles represent the nodes with calculated bootstrap values of 100, 99 and <75%, respectively (1,000 replicates).  Datasets obtained during the present study (races A and B, T1 and T2 (linear and stationary growth phases, respectively)) were compared to the datasets of Guadeloupe race B strain (Condition A-C, supplemented with citric acid as organic carbon source and vitamins (B1, B7 and B12); with A: initial consortium, B: washed culture, C: antibiotics-treated (ciprofloxacin)) 3 . For each sample, 200,000 metagenomic reads were randomly subsampled and subjected to the taxonomic assignment using DIAMOND 4 and MEGAN 1 (minimum 50 reads per taxonomic group). The circle size represents the amount of classified reads for the respective taxonomic group.

Biocoenosis of Botryococcus consortia
The re-processing of the B. braunii Guadeloupe strain metagenome data 3 also shows data qualitatively but not quantitatively similar to the Botryococcus consortia analyzed in this study. The direct comparison of the different metagenome datasets revealed the coinciding presence of the individual genera (especially members of Bacteroidetes, Alpha-and Betaproteobacteria), although massively Biocoenosis of Botryococcus consortia varying in the abundance pattern. The communities differ regarding the presence of the Gram-positive bacterial representatives: while the consortia obtained from our studies harbored the members of Actinobacteria, the members of the phyla Firmicutes were strongly represented in the Guadeloupe strain 3 . This evaluation of the B. braunii consortia under changing cultivation conditions has emphasized that depending on the physiological condition (e.g. supplementation of vitamins and/or antibiotics 3 ), the relative abundance of certain species may widely vary, suggesting the existence of many bacterial taxa with functional redundancy 5 .  Table S1 |

Features of the metagenome assembled genomes (MAGs).
Assembly and annotation characteristics (e.g. Total size, N50, etc.) are listed according to the respective MAG. The completeness as well as the contamination degree of the MAGs was assessed via BUSCO 6,7 (v3.0.). P-and E-Modi refer to prokaryotic MAGs and eukaryotic genome fragments, respectively. The high-quality MAGs were classified via single-copy marker genes, e.g. rpoB, rpoD etc., detected by BUSCO 6,7 . The metagenomic datasets of B. braunii race A and B were mapped against the MAGs, and represented as percentage of mapped reads.

Chapter 1 | Metagenomic survey of the Botryococcus braunii consortia
We profiled four different communities using high-throughput 16S rDNA gene amplicon data  In the race B consortium, less abundant taxa such as Mesorhizobium, Brevundimonas and Hydrogenophaga increased at the expense of Dyadobacter and Devosia. The detected relative levels of both actinobacterial isolates were comparatively low ( Figures S2 and S3). Mycobacterium isolate, obtained from the B. braunii race A community, was almost exclusively propagated therein and increased during stationary growth to 7% of all detected bacterial amplicons ( Figure S3).
Pimelobacter was isolated from the race B consortium, and is present in both communities in equally (≤1%) low abundances which increased slightly during stationary phase.
To test the relationships between B. braunii and the associative consortium, we explored the genetic potential of the abundant representatives of algal-bacterial consortia. We combined metagenome de novo assembly and differential coverage and tetra-nucleotide signature based binning of the B. braunii race A and B samples. This approach resulted in the reconstruction of ten high-quality (completeness >80%, contamination <10%) and ten fragmentary metagenome assembled genomes (MAGs) as well as seven eukaryotic genome fragments (for details see Methods; Table S1). To identify taxa representing the ten high-quality draft MAGs, we constructed a phylogenetic tree built out of a core of 53 highly conserved marker genes per genome ( Figure S4).

Biocoenosis of Botryococcus consortia
All abundant taxa shared by both races were recovered, including the most abundant alphaproteobacterial MAG of the order Rhizobiales, designated DevG (similar to Devosia sp. 66-14, Table S2 and Figure S4). Additionally, MAGs of Dyadobacter and Hydrogenophaga were constructed (DyaG and HydG, similar to Dyadobacter sp. 50-39 and Hydrogenophaga sp. PML113, respectively). Another two alphaproteobacterial MAGs Bre1G and Bre2G of the order Caulobacteriales (similar to Brevundimonas sp. DS20), clustered together within the 16S rDNA amplicon approach ( Figure S3) but were separated through the binning procedure (Table S1) (Table S2), thus underlining the quality of our metagenome assembly and binning approach. The Pimelobacter sp. Bb-B genome sequence resembled the MAG-22, which is only to 24% complete (Table S1).

Chapter 2 | Elucidation of the genetic portfolio of the bacterial community
We performed a KEGG-based quantitative functional assignment of the annotated high-quality MAGs and sequenced genomes by applying the elastic metagenome browser (EMGB, https://emgb.cebitec.uni-bielefeld.de/Bbraunii-bacterial-consortium/). Six of twelve genomes carried genes for bacterial chemotaxis and five of twelve genes for flagella synthesis, hinting at active motility and ability to move towards substrates in the medium (Figure 1b). Botryococcus braunii synthesizes aliphatic long-chain hydrocarbons, which accumulate in large quantities in the extracellular matrix of this species 8 . Seven of twelve genomes carried gene homologs of already characterized hydrocarbon hydroxylases (alkB, ladA, almA and/or cytochrome P450 (CYP), Table   S6) for the initial oxyfunctionalization of long-chain hydrocarbons 9 . While the most abundant consortia members like DevG and DyaG exhibited a very low enzyme number, the more rare Mycobacterium and Pimelobacter genomes contained a large portfolio of hydrocarbon Biocoenosis of Botryococcus consortia monooxygenase/hydroxylase genes, and were the only genomes to carry non-heme membraneassociated monooxygenase alkB genes (Table S6).
Mucilaginous (exo-)polysaccharides as part of the internal fibrillar layer of the B. braunii cell wall represent another prospective carbon source for the bacterial community, consisting mainly of galactose but also of fucose and rhamnose 8,10 . The profiling of the carbohydrate-active enzymes (CAZymes 11 ), encoded by the members of B. braunii communities using dbCAN2 12 metaserver, revealed the presence of several putative enzyme families involved in the complex carbohydrate metabolism (Table S7). Eight out of twelve genomes coded for several carbohydrases for the degradation of poly-and oligosaccharides, generally present in the cell walls of microalgae, and contained genes for chitin disintegration and assimilation (Additional file 2, Table S7). However, six species showed a clear prevalence in the number of enzymes involved in the biosynthesis of carbohydrates (glycosyltransferases) compared to degradation (glycoside hydrolases). Some species like the members of Bacteroidetes contained a large number of CAZyme-encoding genes in their genomes (up to 5.9% of total gene content) with a clear prevalence to hydrolytic activity of complex carbohydrates (Table S7). For instance, the DyaG genome contains more genes encoding for the glycoside hydrolase (GH) families involved in the degradation of complex polymers (cellulose, xylan, chitin), while others, such as the genome of DevG, harbor enzymes acting on oligosaccharides 11 .
Looking at the primary metabolism, all genomes encode genes for processes essential for aerobic respiration (Figure 1b; Table S5). Genes that enable pyruvate fermentation to lactate, acetate and ethanol are also present, showing a versatility of the community members to switch between aerobic and anaerobic metabolisms depending on the oxygen availability. From the perspective of a microalga, the associated bacteria can be providers of micro-and macronutrients but also competitors for limiting nutrients 13 . Based on their observed generic potential, individual species appear to be involved in the acquisition and re-mineralization of macronutrients and in the storage of nitrogen and phosphorous (in form of cyanophycin and Poly-P, Table S6), which benefit the microalga. Eight of twelve genomes encode genes for the synthesis of cyanophycin granule polypeptides (CGPs) 14 , however only four contained genes for the degradation of this C-and Nstorage compound (Table S6). Related to this, all genomes carry genes for the synthesis but not for the degradation of polyphosphate granules (Poly-P), a polymeric reserve with a significant role in the regulation of enzymatic activities, gene expression and stress adaptation processes 15 . Suggesting an adaptation to phosphorous and iron limitation, all genomes contained genes relevant for phosphate starvation (pst system) or siderophore biosynthesis (fhu system).