Isolation and characterization of Flexilinea flocculi gen. nov., sp. nov., a filamentous, anaerobic bacterium belonging to the class Anaerolineae in the phylum Chloroflexi

Abstract

A novel obligately anaerobic bacterium, designated strain TC1^T, was isolated from methanogenic granular sludge in a full-scale mesophilic upflow anaerobic sludge blanket reactor treating high-strength starch-based wastewater. Cells had a multicellular filamentous morphology, stained Gram-negative and were non-motile. The filaments were flexible, generally >100 μm long and 0.3-0.4 μm wide. Growth of the isolate was observed at 25-43 °C (optimum 37 °C) and pH 6.0-8.5 (optimum pH 7.0). Strain TC1^T grew chemo-organotrophically on a range of carbohydrates under anaerobic conditions. Yeast extract was required for growth. The major fermentative end products of glucose, supplemented with yeast extract, were acetate, lactate, succinate, propionate, formate and hydrogen. Co-cultivation with the hydrogenotrophic methanogen <i>Methanospirillum hungatei</i> DSM 864^T enhanced growth of the isolate. The DNA G+C content was determined experimentally to be 42.1 mol%. The major cellular fatty acids were anteiso-C_{15 : 0}, iso-C_{15 : 0} and iso-C_{17 : 0} 3-OH. Based on 16S rRNA gene sequence analysis, strain TC1^T belonged to the class <i>Anaerolineae</i> in the phylum <i>Chloroflexi</i>, in which <i>Ornatilinea apprima</i> P3M-1^T was its closest phylogenetic relative (88.3 % nucleotide identity). Phylogenomic analyses using 38 and 83 single-copy marker genes also supported the novelty of strain TC1^T at least at the genus level. Based on phylogenetic, genomic and phenotypic characteristics, we propose that strain TC1^T represents a novel species of a new genus, for which we suggest the name <i>Flexilinea flocculi</i> gen. nov., sp. nov. The type strain of <i>Flexilinea flocculi</i> is strain TC1^T ( = JCM 30897^T = CGMCC 1.5202^T).