Page 86 - Microbial methane cycling in a warming world From biosphere to atmosphere Michiel H in t Zandt

Chapter 4. Robustness of coal microbial community after nutrient amendment
annotated against a Geobacter reference data set containing nine annotated genomes. Contig sequences were uploaded to the KEGG Automated Annotation Server (KAAS) (Moriya et al. 2007) and analyzed using 40 most related reference organisms. Specific protein sequence data sets for metal reduction, complex organic compounds degradation and genus specific genes were manually selected and downloaded from the UniProt database (http://www.uniprot.org/) (The UniProt Consortium 2014). For the analysis of the fungal community, fungal peroxidase databases were made for lignin, manganese and versatile peroxidases by downloading nucleotide data from the NCBI database, filtering for fungal sequences ≤ 100,000 bp. Reference databases were imported in CLC and mapped against the genomic data sets using the CLC BLASTX tool using the standard parameters: expect: 10.0, match: 2, mismatch: -3, gap costs existence: 5, gap cost extension: 2. Artemis 16.0.0 was used for visualization of the annotation results (Rutherford et al. 2000). Methanogen and methanotroph functional gene analyses were performed using McrA, MmoX and PmoA/AmoA protein sequence databases and a non- redundant reference database (Claudia Lüke, Radboud University, personal communication). Analysis of trimmed reads was performed using the BLASTX (BLAST 2.2.25+) algorithm with E-value 0.0001 extracting first hits as target sequence. Functional gene and non-redundant BLAST results with a bitscore >125 were merged. All reads had a BLAST score ratio of > 0.8 (Lüke et al. 2016). Only McrA analysis resulted in significant hits.
Nucleotide sequence accession numbers
All sequencing data were submitted to the GenBank databases under the BioProject PRJNA321375. The assembled Ion Torrent data set from this study was submitted as Sequence Read Archive study under accession number SRP074879 (run SRR5328925). The amplicon data were submitted as SAMN07344736 (17 months archaeal), SAMN07344812 (17 months bacterial), SAMN07344862 (25 months archaeal) and SAMN07344863 (25 months bacterial).
Results and Discussion
The two experimental coal wells are located at the Lithgow State Coal Mine in the Western Coal Fields of New South Wales, Australia. The coal bearing layers are located 80 m below ground level. About 2 m2 of coal is continuously exposed to the groundwater that has a volume
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