Page 61 - Microbial methane cycling in a warming world From biosphere to atmosphere Michiel H in t Zandt

reductase [McrA], particulate methane monooxygenase [PmoA] and soluble methane monooxygenase [MmoX]), and acetate metabolism (acetate kinase [AckA], acetyl-coa synthetase [ACS], and carbon monoxide dehydrogenase [CODH]) were selected and downloaded from the NCBI (see the supplemental material for NCBI database generation). Analysis of trimmed reads was performed using the BLASTX (BLAST 2.2.25+) algorithm with an E-value of 0.0001, extracting first hits as a target sequence. Output sequences were manually checked and filtered on bitscore to obtain true hits. For McrA, the bitscore was set to ≥100, for PmoA and MmoX no true sequences were obtained, for AckA the bitscore was set to ≥80, for ACS the bitscore was set to ≥80, and for CODH no true target hits were obtained. For each draft genome bin, the selected genes were also obtained. For the final species identification, the bitscore-filtered reads were blasted against the NCBI non-redundant protein database (13 December 2018) using CLC Genomics Workbench 11 with custom settings Expect 10.0, Word size 3, Matrix BLOSUM62 and gap costs Existence 11, Extension 1. To extract reads that matched the 16S rRNA gene used for taxonomic classification, all reads were mapped against the SILVA SSU 132 data set using CLC Genomics Workbench v11. Length and similarity fraction were set to 50% and 70%, respectively, and values for mismatch, insertion and deletion cost to 2, 3 and 3, resulting in a total of 63,478 mapped reads (0.25% of total). The extracted 16S rRNA gene reads were also used for de novo assembly. 16S rRNA gene contigs were assessed using nucleotide BLAST against the NCBI GenBank as described by in ‘t Zandt et al. (2018).
Nucleotide sequence accession numbers
MiSeq sequencing data were deposited in the European Nucleotide Archive under accession number ERP008629.
Results and Discussion
We studied 4-m-deep sheet piles at a pumping station in Gouderak, the Netherlands, that were removed after 50 years of service. Natural CPLs were observed on the surface of the sheet piles. These deposit layers have the potential to protect iron sheet piles from corrosion, resulting in a longer life span, with corresponding economic and environmental benefits. Therefore, we
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