Base Mine Lake (BML) is the first full-scale demonstration end pit lake for the oil sands mining industry in Canada. We examined aerobic methanotrophic bacteria over all seasons for five years in this dimictic lake. Methanotrophs comprised up to 58% of all bacterial reads in 16S rRNA gene amplicon sequencing analyses (median 2.8%), and up to 2.7 × 10 4 cells mL −1 of water (median 0.5 × 10 3 ) based on qPCR of pmoA genes. Methanotrophic activity and populations in the lake water were highest during fall turnover, and remained high through the winter ice-covered period into spring turnover. They declined during summer stratification, especially in the epilimnion. Three methanotroph genera ( Methylobacter , Methylovulum , and Methyloparacoccus ) cycled seasonally, based on both relative and absolute abundance measurements. Methylobacter and Methylovulum populations peaked in winter/spring, when methane oxidation activity was psychrophilic. Methyloparacoccus populations increased in the water column through summer and fall, when methane oxidation was mesophilic, and also predominated in the underlying tailings sediment. Other, less abundant genera grew primarily during summer, possibly due to distinct CH 4 /O 2 microniches created during thermal stratification. These data are consistent with temporal and spatial niche differentiation based on temperature, CH 4 and O 2 . This pit lake displays methane cycling and methanotroph population dynamics similar to natural boreal lakes. Importance statement: The study examined methanotrophic bacteria in an industrial end pit lake, combining molecular DNA methods (both quantitative and descriptive) with biogeochemical measurements. The lake was sampled over 5 years, in all four seasons, as often as weekly, and included sub-ice samples. The resulting multi-season and multi-year dataset is unique in its size and intensity, and allowed us to document clear and consistent seasonal patterns of growth and decline of three methanotroph genera ( Methylobacter , Methylovulum , and Methyloparacoccus ). Laboratory experiments suggested that one major control of this succession was niche partitioning based on temperature. The study helps to understand microbial dynamics in engineered end-pit lakes, but we propose that the dynamics are typical of boreal stratified lakes, and widely applicable in microbial ecology and limnology. Methane oxidising bacteria are important model organisms in microbial ecology, and have implications for global climate change.

GAL08 are bacteria belonging to an uncultivated phylogenetic cluster within the phylum Acidobacteria. We detected a natural population of the GAL08 clade in sediment from a pH-neutral hot spring located in British Columbia, Canada. To shed light on the abundance and genomic potential of this clade, we collected and analyzed hot spring sediment samples over a temperature range of 24.2–79.8°C. Illumina sequencing of 16S rRNA gene amplicons and qPCR using a primer set developed specifically to detect the GAL08 16S rRNA gene revealed that absolute and relative abundances of GAL08 peaked at 65°C along three temperature gradients. Analysis of sediment collected over multiple years and locations revealed that the GAL08 group was consistently a dominant clade, comprising up to 29.2% of the microbial community based on relative read abundance and up to 4.7 × 105 16S rRNA gene copy numbers per gram of sediment based on qPCR. Using a medium quality threshold, 25 single amplified genomes (SAGs) representing these bacteria were generated from samples taken at 65 and 77°C, and seven metagenome-assembled genomes (MAGs) were reconstructed from samples collected at 45–77°C. Based on average nucleotide identity (ANI), these SAGs and MAGs represented three separate species, with an estimated average genome size of 3.17 Mb and GC content of 62.8%. Phylogenetic trees constructed from 16S rRNA gene sequences and a set of 56 concatenated phylogenetic marker genes both placed the three GAL08 bacteria as a distinct subgroup of the phylum Acidobacteria, representing a candidate order (Ca. Frugalibacteriales) within the class Blastocatellia. Metabolic reconstructions from genome data predicted a heterotrophic metabolism, with potential capability for aerobic respiration, as well as incomplete denitrification and fermentation. In laboratory cultivation efforts, GAL08 counts based on qPCR declined rapidly under atmospheric levels of oxygen but increased slightly at 1% (v/v) O2, suggesting a microaerophilic lifestyle.