Identifying microbial functional guilds performing cryptic organotrophic and lithotrophic redox cycles in anaerobic granular biofilms

Abstract

Granular biofilms used in anaerobic digester systems contain diverse microbial populations that interact to hydrolyze organic matter and produce methane within controlled environments. Prior research investigated the feasibility of utilizing granular biofilms obtained from an anaerobic digester to remove nitrate without the addition of exogenous electron donors. These granules possessed a unique structure of alternating light and dark iron sulfide and pyrite rich layers that potentially served as both an electron source and sink, linking carbon, nitrogen, sulfur, and iron cycles. To characterize the functional roles of diverse microbial populations enriched within these layered biofilms, we analyzed metagenomes obtained from three different granules. Comparisons between the functional gene content of forty metagenome assembled genomes (MAGs) identified phylogenetically cohesive functional guilds. Each of these functional MAG clusters was assigned to specific steps in anaerobic digestion (hydrolysis, acidogenesis, acetogenesis, and methanogenesis) and anaerobic respiration (denitrification and sulfate reduction). Comparisons with metagenomes derived from a variety of natural and engineered ecosystems confirmed that the enriched denitrifying bacteria were similar to populations typically found in wetlands and biological nitrogen removal systems. Analysis of read alignments to individual genes within the forty MAGs identified conserved genomic features that were representative of the functions that distinguished functional guilds. Overall, this research illustrates the utility of functional based classification of microorganisms for characterizing ecosystem functions and highlights the potential application of engineered ecosystems to serve as experimental models for complex natural ecosystems.

Fig 1. Layered structure of granular biofilms.

Anaerobic granule sliced showing alternating light and dark layers. Granule sizes were typically in the 3 to 5 mm diameter range.

Fig 2. Functional and taxonomic characterization of granular biofilms.

(A left to right) The dendrogram presents clustering based on Pearson correlation of normalized KEGG ortholog (KO) annotation counts for 40 MAGs followed by genomic characteristics and taxonomic assignments. The heatmap depicts CAZyme counts (log transformed, normalized, and scaled to a range of −2 to 2) for each category: AA-auxiliary activities, GT-glycosyl transferase, CBM-carbohydrate binding motif, PL-polysaccharide lyase, GH-glycoside hydrolase, and CE-carbohydrate esterase. The grid indicates the presence of specific functional genes, hydrogenase (hydAB), methyl coenzyme M reductase (mcrA), dissimilatory sulfite reductase (dsrAB), formate dependent nitrite reductase (nrfAH), periplasmic nitrate reductase complex (napAB), dissimilatory nitrate reductase (narGH), nitrite reductases (nirK and nirS), nitric oxide reductase (norB), and nitrous oxide reductase (nosZ). The color of each gene column corresponds to the redox processes (i-vii) in panel C. The taxonomic assignment for each MAG is designated by the same colors as in panel B. (B) Stacked bar-chart representing the microbial community structure based on RPKM values assessed for each recovered bin for each of the three Illumina sequence libraries obtained for each granule, columns A, B, and C. Each bar includes the summed RPKM values for all bins for each assigned taxonomy. “Other” includes bins assigned to low abundance taxa, taxa without a representative high-quality MAG, and all un-binned contigs. (C) Diagram of potential biologically mediated redox processes (i-vii) occurring within the granules.

Fig 3. Metagenomic comparisons.

Heatmap presenting log normalized and scaled (−2 to 2) RPKM values obtained by mapping reads from 31 metagenomes and the three granule metagenome libraries (A, B, and C from this study) represented by each column against the 40 high MAGs, each row. Clustering of columns was based on pairwise correlation scores between each sample’s set of bin RPKM values. The order and taxonomy of MAGs in each row is consistent with Fig 1. Heatmap columns for compared metagenomes are numbered and the corresponding descriptions are listed in S5 Table in S1 File.

Fig 4. Identification and functional clustering of conserved genes.

(A) Clustering of 40 MAGs based on the reduced set of KOs identified as conserved among any of the MAGs. Taxonomy is indicated by the legend. (B) Percent of genes within each genome identified as having significant read mapping (blue bars) and the percent of genes in each genome identified as significant but not having any annotation assignment from KEGG, eggNOG, or CAZyme database searches (cyan bars). (C) The left panel histogram depicts the distribution of MAGs within each range of genome proportion identified as significant (1% width bins, blue bars). The right panel histogram presents the distribution of MAGs based on percent of genes identified as significant but not annotated (0.25% width bins, cyan bars).

Fig 5. Granular biofilms as model systems for complex soil ecosystems.

The granular biofilms examined in this study possess microbial functional guilds, clusters 1-8, that perform similar functions as in the more complex microbial communities of natural soil aggregates.