Metagenome-assembled genomes of the methanogenic enrichment obtained from drilling fluid wastes stored in permafrost

TL;DR

This paper identifies methanogenic microbes in drilling fluid waste stored in permafrost.

Contribution

The study provides metagenome-assembled genomes from a methanogenic enrichment in permafrost drilling waste.

Findings

The enrichment contains Methanosarcina, Methanobacterium, Proteinifillum, and Synergistetes.

The study focuses on methanogenic microbes from a specific permafrost drilling site.

Abstract

We studied methanogenic enrichment (Kha) from spent drilling fluid stored in permafrost, Kharasavey (71°10′50″N 66°51′50″E) gas field located in the western part of the Yamal Peninsula. The metagenome-assembled genomes showed that Kha consists of representatives of Methanosarcina, Methanobacterium, Proteinifillum, and Synergistetes genera.

Tables

TABLE 1. Description of MAGs obtained during the study

Genome feature	MAGs
	IBPM_KMB_001	IBPM_KMB_002	VO1	IBPM_KMB_004
Closest genome (GTDBtk data)	GW-Synergistetes-1, MAGGCA_002839185.1	Proteiniphilum saccharofermentans M3/6T GCF_900095135.1	Methanosarcina sp. 2.H.T.1A.6, MAG GCF_000979455.1	Methanobacterium sp. Maddingley MBC34, MAG GCA_000309865.1
OrthoANI with closest genome (%)	91.06	95.34	84.56	86.26
Completeness/contamination (CheckM)	100/0	99.45/0	99.84/0.65	98.93/0.8
Genome coverage	166 ± 33	603 ± 224	383 ± 146	40 ± 11
Read abundance (%)	2,457,644 (6.94)	15,325,445 (43.29)	11,055,016 (31.23)	679,997 (1.92)
Genome length (bp)	2,214,899	3,800,659	4,322,233	2,538,249
Scaffolds	17	59	144	9
N 50	396,354	102,999	44,010	489,195
L 50	3	13	30	3
GC content (%)	46.8	43.8	41.6	39.2
Genes (total)	2,065	3,039	3,630	2,505
CDSa (total)	2,016	2,993	3,571	2,453
Genes (coding)	2,009	2,957	3,518	2,448
tRNA	45	44	57	46
16S, 5S, 23S rRNA	0, 1, 0	–	–	2, 1, 1

Full text

ANNOUNCEMENT

The drilling fluid (DF) (70 g) sample was collected using a sterile sampler into a sterile container and transported to the laboratory at –20°C. After a half-year anaerobic incubation in a 150-mL glass flask at room temperature, 1 mL of fermented DF was transferred in 20 mL of DSMZ 141 medium (1) with CO_2_:H_2_ (80:20) and methanol as substrates and was anaerobically incubated at 20°C for a month. After four transfers in the same conditions, we received the stable methanogenic enrichment Kha.

Genomic DNA preparation and sequencing were performed by BioSpark Company (Troitsk, Russia). Genomic DNA from Kha was isolated with the FastDNA spin kit (MP Biomedicals, USA) using the column method and deposition on silica gel. The paired-end libraries were synthesized using KAPA HyperPlus kits (Kapa Biosystems, USA) in accordance with the manufacturer’s recommendations. Sequencing was performed on the Illumina NovaSeq 6000 platform, and a paired-end library with a total of 17,702,025 reads and a read length of 150 bp with a mean quality of 35 was obtained.

To obtain metagenome-assembled genomes (MAGs), the Kbase online platform with appropriate tools was used (2). The quality of the reads was assessed using the FastQC v.0.12.1 program (3). Reads were processed in Trimmomatic (4) with the parameters “HEADCROP = 15, MINLEN = 50” and removing adapters from the TruSeq3-PE-2 set. Contigs and scaffolds were assembled from paired libraries using MetaSPAdes v.3.15.3 with default parameters (5). Contig binning was performed using the CONCOCT v.1.1 (6) with subsequent optimization of the result in DAS Tool v.1.1.2 with BLAST gene identification (7). The completeness of the obtained genomes was assessed using CheckM v.1.0.18 (8), and taxonomic affiliation was determined in GTDB-tk v.2.3.2 (9). The orthoANI calculation was made using the online service https://www.ezbiocloud.net/tools/ani. Alignment of reads to genomes and determination of coverage and the number of aligned reads were carried out using bowtie2 v. 2.3.2 (10). Default parameters were used for all software unless otherwise noted. Rapid annotation of genomes was carried out with the CBI Prokaryotic Genome Annotation Pipeline (11).

As a result of the work, four MAGs were obtained, two of which belonged to methanogenic archaea (Methanosarcina and Metanobacterium genera), and two belonged to Bacteria (Table 1).

Of the resulting assemblies, one (IBPM_KMB_004) was a high-quality MAG (completeness was more than 90%; contamination was less than 5%; 16S, 5S, 23S, and at least 16 tRNA were present). The remaining three drafts were close to high-quality MAGs, except for the absence of 16S/5S/23S rRNA in three representatives (12). All MAGs demonstrated a large difference in the orthoANI coefficient with the closest relatives and may belong to new bacterial and archaeal taxa.