The development of reliable, mixed-culture biotechnological processes hinges on understanding how microbial ecosystems respond to disturbances. Here we reveal extensive phenotypic plasticity and niche complementarity in oleaginous microbial populations from a biological wastewater treatment plant. We perform meta-omics analyses (metagenomics, metatranscriptomics, metaproteomics and metabolomics) on in situ samples over 14 months at weekly intervals. Based on 1,364 de novo metagenome-assembled genomes, we uncover four distinct fundamental niche types. Throughout the time-series, we observe a major, transient shift in community structure, coinciding with substrate availability changes. Functional omics data reveals extensive variation in gene expression and substrate usage amongst community members. Ex situ bioreactor experiments confirm that responses occur within five hours of a pulse disturbance, demonstrating rapid adaptation by specific populations. Our results show that community resistance and resilience are a function of phenotypic plasticity and niche complementarity, and set the foundation for future ecological engineering efforts.

Short-read sequencing technologies have long been the work-horse of microbiome analysis. Continuing technological advances are making the application of long-read sequencing to metagenomic samples increasingly feasible. We demonstrate that whole bacterial chromosomes can be obtained from an enriched community, by application of MinION sequencing to a sample from an EBPR bioreactor, producing 6 Gb of sequence that assembles into multiple closed bacterial chromosomes. We provide a simple pipeline for processing such data, which includes a new approach to correcting erroneous frame-shifts. Advances in long-read sequencing technology and corresponding algorithms will allow the routine extraction of whole chromosomes from environmental samples, providing a more detailed picture of individual members of a microbiome.

Management of phosphorus discharge from human waste is essential for the control of eutrophication in surface waters. Enhanced biological phosphorus removal (EBPR) is a sustainable, efficient way of removing phosphorus from waste water without employing chemical precipitation, but is assumed unachievable in tropical temperatures due to conditions that favour glycogen accumulating organisms (GAOs) over polyphosphate accumulating organisms (PAOs). Here, we show these assumptions are unfounded by studying comparative community dynamics in a full-scale plant following systematic perturbation of operational conditions, which modified community abundance, function and physicochemical state. A statistically significant increase in the relative abundance of the PAO Accumulibacter was associated with improved EBPR activity. GAO relative abundance also increased, challenging the assumption of competition. An Accumulibacter bin-genome was identified from a whole community metagenomic survey, and comparative analysis against extant Accumulibacter genomes suggests a close relationship to Type II. Analysis of the associated metatranscriptome data revealed that genes encoding proteins involved in the tricarboxylic acid cycle and glycolysis pathways were highly expressed, consistent with metabolic modelling results. Our findings show that tropical EBPR is indeed possible, highlight the translational potential of studying competition dynamics in full-scale waste water communities and carry implications for plant design in tropical regions.

New long read sequencing technologies offer huge potential for effective recovery of complete, closed genomes from complex microbial communities. Using long read data (ONT MinION) obtained from an ensemble of activated sludge enrichment bioreactors we recover 22 closed or complete genomes of community members, including several species known to play key functional roles in wastewater bioprocesses, specifically microbes known to exhibit the polyphosphate- and glycogen-accumulating organism phenotypes (namelyCandidatusAccumulibacter andDechloromonas, andMicropruina,DefluviicoccusandCandidatusContendobacter, respectively), and filamentous bacteria (Thiothrix) associated with the formation and stability of activated sludge flocs. Additionally we demonstrate the recovery of close to 100 circularised plasmids, phages and small microbial genomes from these microbial communities using long read assembled sequence. We describe methods for validating long read assembled genomes using their counterpart short read metagenome-assembled genomes, and assess the influence of different correction procedures on genome quality and predicted gene quality. Our findings establish the feasibility of performing long read metagenome-assembled genome recovery for both chromosomal and non-chromosomal replicons, and demonstrate the value of parallel sampling of moderately complex enrichment communities to obtaining high quality reference genomes of key functional species relevant for wastewater bioprocesses.