Metagenomes obtained by "deep sequencing" - what do they tell about the EBPR communities? Mads Albertsen1, Aaron M. Saunders1, Kåre L. Nielsen1 and Per H. Nielsen1 1 Department of Biotechnology, Chemistry and Environmental Engineering, Aalborg University, Aalborg, Denmark Presenting Author: Mads Albertsen Keywords: Metagenomics; Accumulibacter; Micro-diversity; Enhanced Biological Phosphorus Removal Introduction Metagenomics, or environmental genomics, provides comprehensive information about the entire microbial community of a certain ecosystem, e.g. a wastewater treatment plant. So far, metagenomic analyses have been hampered by high costs and high level of expertise needed to conduct the investigations, but it is changing now with development of new technologies allowing analyses of billions of DNA sequences (deep-sequencing) and user-friendly pipelines for analyses of the huge data sets. Analyses of metagenomes can give extensive information about the identity of microbes in the community as well as their functional potential (Kuczynski et al., 2011). Metagenomics can also be regarded as the blueprint needed to conduct the next tier of studies of gene and protein (transcriptomics and proteomics) providing information about the function of the microbes in the system. The combination of metagenomics, transcriptomics and proteomics will bring us closer to a systems microbiology perspective of a certain microbial community and will aid in the goal of establishing predictive models. Recently we presented the first metagenome of a full-scale wastewater treatment plant carrying out nitrification/denitrification and enhanced biological phosphorus removal (EBPR) (Albertsen et al., 2011). Such studies on full-scale plants are needed to improve the understanding of key microorganisms in the EBPR process (e.g., the polyphosphate accumulating bacteria, PAO) and the entire community in order to improve the process for efficient P-removal and P-recovery (Nielsen et al., 2010; 2011). The aim of this study was to compare metagenomes from two full-scale EBPR plants to other environments and to investigate in detail similarities and differences between the two EBPR communities. Material and Methods DNA extraction from activated sludge from the EBPR wastewater treatment plants Aalborg East and West and the further metagenomic sequencing, assembly and annotation were largely conducted as described in Albertsen et al., (2011). Results and Discussion We sequenced two metagenomes from Aalborg East and West EBPR wastewater treatment plants at a depth of 12 and 8 Gb using Illumina short read sequencing. The EBPR plants form a distinct group when compared to metagenomes from a wide range of environments, both on phylogenetic and functional level (Fig. 1). Even though the samples were taken at different times of the year (August vs. December) and from different EBPR plants, they cluster tightly, which may be attributed to the wide range of selection pressures acting on the EBPR communities. These results confirm the findings of a core microbial community using quantitative fluorescence in situ hybridization (qFISH) and other techniques (Nielsen et al., 2010; 2011). Through the use of qFISH probes we investigated the micro-diversity of the key PAO Accumulibacter (clade I and II) in the two EBPR plants. In Aalborg East clade I was estimated to 1.6% and II to 1.3% of the total population, and 1.5% and 1.1% in West. As a reference genome exists for clade IIA we used the raw metagenome reads to estimate the Accumulibacter micro-diversity in the metagenomes. This revealed a much greater micro-diversity than observed through qFISH - also between the two metagenomes. Despite the large diversity the PAO core functions seemed conserved between the different Accumulibacter species. In addition, there seemed to be a high selection pressure from viruses (phages) acting on the different Accumulibacter populations. Conclusions The improved resolution of deep metagenomics enables insights of both community function and micro-diversity. We showed that the complex selection pressures in EBPR plants seem to create a tightly controlled core gene pool, although significant micro-diversity exists. The dynamics of micro-diversity at genome level and the implications for stable plant operation and P-removal will be an interesting question to investigate further. One current limitation for application of metagenomics and metatranscriptomics on a systems level is the need of more reference genomes that are closely related to the species in the EBPR plants. Currently only a handful of relevant genomes are available, however the limited core species in the EBPR process and the rapid improvement of single cell genomics makes us confident that we soon will be able to conduct comprehensive systems microbiology investigations in EBPR plants. References Albertsen M., Hansen L.B.S., Saunders A.M., Nielsen P.H., and Nielsen K.L. (2011). A metagenome of a full-scale microbial community carrying out enhanced biological phosphorus removal. ISME journal, ePub ahead of print, doi :10.1038/ismej.2011.176. Nielsen P.H., Mielczarek A.T., Kragelund C., Nielsen J.L., Saunders A.M., Kong Y., et al. (2010). A conceptual ecosystem model of microbial communities in enhanced biological phosphorus removal plants. Water Res 44, 5070-5088. Nielsen P.H., Saunders A.M, Hansen A.A, Larsen P., Nielsen J.L (2011) Microbial communities involved in enhanced biological phosphorus removal from wastewater - a model system in environmental biotechnology. 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