Solid-state anaerobic co-digestion of food waste, rice straw, and rice husk supplemented with cattle digesta under thermophilic conditions

Shimizu, Hiroyuki; Matsuura, Norihisa; Gu, Tingting; Tsuritani, Takashi; Okamoto, Masanori; Yamamoto-Ikemoto, Ryoko

doi:10.1007/s10163-022-01484-9

Cited by 3 publications

(1 citation statement)

References 35 publications

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“…In natural systems, such as soil (Pérez Castro et al, 2019; Hao et al, 2021) and water (Morrison et al, 2017; Teachey et al, 2019), 16S rRNA gene amplicon sequencing analysis is used to confirm the effects of environmental factors on the composition and diversity of microorganisms. Microbial analysis is also used to assess and monitor the composition of microbial communities in artificial ecosystems, such as methane fermentation (Shimizu et al, 2022; Yu et al, 2018) and biological water treatment processes (Liang et al, 2020; Matsuura et al, 2021).…”

Section: Introductionmentioning

confidence: 99%

A quantitative sequencing method using synthetic internal standards including functional and phylogenetic marker genes

Koike

Honda

Aoki

et al. 2023

Environ Microbiol Rep

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The method of spiking synthetic internal standard genes (ISGs) to samples for amplicon sequencing, generating sequences and converting absolute gene numbers from read counts has been used only for phylogenetic markers and has not been applied to functional markers. In this study, we developed ISGs, including gene sequences of the 16S rRNA, pmoA, encoding a subunit of particulate methane monooxygenase and amoA, encoding a subunit of ammonia monooxygenase. We added ISGs to the samples, amplified the target genes and performed amplicon sequencing. For the mock community, the copy numbers converted from read counts using ISGs were equivalent to those obtained by the quantitative real‐time polymerase chain reaction (4.0 × 104 versus 4.1 × 104 and 3.0 × 103 versus 4.0 × 103 copies μL‐DNA−1 for 16S rRNA and pmoA genes, respectively), but we also identified underestimation, possibly due to primer coverage (7.8 × 102 versus 3.7 × 103 μL‐DNA−1 for amoA gene). We then applied this method to environmental samples and analysed phylogeny, functional diversity and absolute quantities. One Methylocystis population was most abundant in the sludge samples [16S rRNA gene (3.8 × 109 copies g−1) and the pmoA gene (2.3 × 109 copies g−1)] and were potentially interrelated. This study demonstrates that ISG spiking is useful for evaluating sequencing data processing and quantifying functional markers.

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