Metagenomic and Genomic Sequences from a Methanogenic Benzene-Degrading Consortium

ABSTRACT

Draft and complete metagenome assembled genomes (MAGs) were created from multiple metagenomic assemblies of DGG-B, a strictly anaerobic, stable mixed microbial consortium that degrades benzene completely to methane and CO₂. Our objective was to obtain closed genome sequences of benzene-fermenting bacteria to enable the elucidation of their elusive anaerobic benzene degradation pathway.

ANNOUNCEMENT

DGG-B refers to a scaled-up lineage (>100 L) of a methanogenic benzene-degrading enrichment culture called “OR” derived from microcosms constructed in 1995 with sediments from an oil refinery (1, 2). OR and DGG-B cultures are grown routinely in a minimal medium amended monthly with 25 mg/L benzene. In 2010, preliminary metagenomic sequencing (3) indicated that OR was composed primarily of two uncultured strains of benzene-fermenting Deltaproteobacteria (now Desulfobacterota), namely, ORM2a and ORM2b; a candidate phylum bacterium (OD1, later reclassified as Candidatus Nealsonbacteria); and two methanogens (Methanothrix and Methanoregula). OR and DGG-B also contain >100 low-abundance microorganisms that are not well characterized (2–4). To learn more about these complex microbial communities and to shed light on their unknown mechanism of benzene degradation, metagenomic assemblies were used to reconstruct draft and complete genome sequences.

DNA was extracted from aliquots of two subcultures of DGG-B in 2016 (DGG1A, 15 mL) and in 2017 (DGG0, 200 mL) using the Qiagen DNeasy PowerSoil kit. At the time of sampling, the total volumes of DGG1A and DGG0 were 15.9 L and 9.0 L, respectively, and degraded benzene at a rate of 0.8 to 1.5 mg/L/day. Metagenomic sequencing was performed using long-read (PacBio) and/or short-read (Illumina) methods summarized in Fig. 1. Following the metagenomics workflow illustrated in Fig. 1, raw reads were trimmed and filtered (5), assembled into contigs (6–8), and binned (9–12). Default parameters were used for all software; for MEGAHIT assemblies, we also used the “sensitive” preset option. Bins with a pairwise average nucleotide identity of ≥99%, genome completeness of >50%, and contamination of <25% were compared and dereplicated (13). Taxonomy was assigned to the resulting draft MAGs using the GTDB-tk tool kit (14). Three draft MAGs were selected for further refinement (Table 1). In ABySS v.1.3.7 (15), new assemblies were created using a coverage of ≥80 (for ORM2a only) and/or k-mer of 96. ABySS contigs were then mapped onto the existing contigs, and gaps were resolved using a perl script published in Text S1 of Tang et al. (16). Finished genomes were polished by read mapping raw reads onto circular genomes using the Bowtie v.7.2.1 plugin within Geneious v.8.1.8 (17). Assembly information and MAG statistics were generated in CheckM v.1.0.18 (18) and using the anvi-summarize command within Anvi’o v.6.2 (19).

FIG 1.

Metagenomics workflow used for (hybrid) assembly, binning, and curation of draft DGG-B MAGs. Default parameters were used for all software unless otherwise specified. PE, paired-end.

TABLE 1.

Summary of two metagenomes and three closed MAGs reported in this study^a

Name	Genome affiliation (GTDB-tk)	Genome affiliation (NCBI)	(Meta) genome length (Mb)	No. of contigs	GC content (%)	Avg coverage depth (×)	Genome completeness (%)	Genome contamination (%)	GenBank accession no.	IMG ID
DGG-1A metagenome	N/Ab	N/A	256	191,164	51.2	56	N/A	N/A	JARDUI000000000	3300019861
DGG0 metagenome (PE + PacBio hybrid assembly)	N/A	N/A	231	355,455	49.6	28	N/A	N/A	JARDUJ000000000	3300028923
Desulfobacterota candidate ORM2a	Desulfobacterota; BSN033; UBA8473	Desulfobacterota	3.30	1	44.1	1,765 (DGG1A); 979 (DGG0)	100	0	CP113000.1	2795385393
Candidatus Nealsonbacteria DGGOD1	Patescibacteria; Paceibacterales; UBA5738	Patescibacteria; Candidatus Nealsonbacteria	1.16	1	45.8	278 (DGG1A); 113 (DGG0)	100	0	CP092821.1	2791354853
Methanoregula sp. strain DGG0	Methanoregulaceae	Methanoregula	2.17	1	52.0	169 (DGG1A); 78 (DGG0)	100	0	CP112999.1	2799112206

^aA table summarizing all other (draft) MAGs created in this study is available in figshare (https://doi.org/10.6084/m9.figshare.21663302.v1).

^bN/A, not available.

Seventy-four draft MAGs were recovered from DGG-B metagenomes, representing 71 to 73% of total microorganisms as determined by relative abundance estimates in Anvi’o (19). Further refinement enabled us to close the genomes of ORM2a, a Candidatus Nealsonbacteria (designated DGGOD1a), and a Methanoregula sp. strain (Table 1). Other MAGs recovered included members of the Bacteroidales (n = 10), Anaerolineales (n = 6), Syntrophales (n = 5), and Methanothrix (n = 8). Our laboratory is currently analyzing the genomes of ORM2a (C. R. A. Toth, O. Molenda, C. Nesbo, F. Luo, C. Devine, R. Flick, and E. A. Edwards, unpublished data) and Ca. Nealsonbacteria (20).

Data availability.

Nucleotide accession numbers for reported metagenomes and refined MAGs are provided in Table 1. FASTA files and relevant statistics for all other draft MAGs are available in FigShare (https://doi.org/10.6084/m9.figshare.21663302.v1). An assembly of the original OR metagenome and a draft ORM2 bin are also available in the US DOE Joint Genome Institute Integrated Microbial Genomes (IMG) system (identifiers [IDs] 3300001389 and 2739367767, respectively).