Jonas, Lauren

ABSTRACT The functional roles of bacterial symbionts associated with microalgae remain understudied despite the importance of microalgae in biotechnology and environmental microbiology. 16S rRNA gene sequencing was conducted to analyze bacterial communities associated with two microalgae optimized for growth with flue gas containing 5%–10% CO 2 . Two dominant bacteria with no taxonomic classification beyond the class level (Paceibacteria) were discovered repeatedly in the most productive algal cultures. Long-read metagenomic sequencing was conducted to yield high-quality metagenomes, from which two novel species were discovered under the Seqcode (seqco.de/r:ywe1blo2), Phycocordibacter aenigmaticus gen. nov. sp. nov. and Minusculum obligatum gen. nov. sp. nov. The genus Phycocordibacter gen. nov. was proposed as the nomenclatural type of the family Phycocordibacteraceae fam. nov. and the order Phycocordibacterales ord. nov. Both bacteria possessed features typical of Patescibacteria such as reduced genomes (<800 kbp), lack of complete glycolysis and tricarboxylic acid (TCA) cycle pathways, and inability to synthesize amino acids. Instead, they rely on the reductive pentose phosphate pathway (Calvin cycle) for essential biosynthesis and redox balance. P. aenigmaticus may also rely on elemental sulfur oxidation ( sdo ), partial nitrite reduction ( nirK ), and sulfur-related amino acid metabolism (SAMe → SAH). Both bacteria were found in high relative abundance in cultures of Tetradesmus obliquus HTB1 (freshwater) and Nannochloropsis oceanica IMET1 (marine), suggesting a tight association with microalgae in various environments. The absence of full metabolic pathways for energy production suggests extreme metabolic limitations and obligate symbiosis, most likely with other bacteria associated with the microalgae. IMPORTANCE To our knowledge, this is the first report of Patescibacteria as dominant bacteria associated with microalgae or within a biologically mediated carbon capture system. Two novel Patescibacteria were found in two ecologically distinct microalgal cultures (one freshwater strain and one marine) regardless of whether the cultures were bubbled with air, 5% CO 2 , or 10% CO 2 . This unexpected and unprecedented dominance led to long-read sequencing and the assembly of high-quality metagenomes for both Patescibacteria, as well as five other bacteria in the system. The discovery of two novel species belonging to two novel genera, one novel family, and one novel order has enabled us to fill in gaps of a major, uncharacterized branch within the bacterial tree of life. Additionally, the extreme gene loss found in both Patescibacteria, Phycocordibacter aenigmaticus and Minusculum obligatum , contributes knowledge to a rapidly advancing body of research on the scavenging metabolic nature of this enigmatic and largely unclassified phylum.