Soil microbial community and structure
Soil microbial community structures were analyzed with PLFAs
(phospholipid fatty acids) methods (Bossio & Scow 1998; Zhang et
al. 2016), accounting for active microbes which were directly related
to soil ecosystem functioning. Briefly, 8-g freeze-dried soil sample was
added to 23 mL extraction solution containing chloroform: methanol:
phosphate buffer (1: 2: 0.8 v/v/v). These extracted polar lipids were
separated from neutral and glycolipids on a silica column (Cleanert
Silica cartridge, 500 mg 6 mol L-1, Agela Technologies
Inc.). After mild alkaline methanolysis, polar lipids were converted to
fatty acid methyl esters. The individual fatty acid methyl esters in the
sample were analyzed by an Agilent 7890A gas chromatograph (Santa Clara,
CA, USA) with MIDI peak identification software. The concentrations of
each PLFAs were calculated based on their ratio to the 19:0 internal
standard (5 mg mL-1). Total extractable PLFAs were
used as a proxy for soil microbial biomass.
Meanwhile, for each soil sample, total genomic DNA was extracted from
500 mg fresh soil using a MO BIO DNA extraction kit (MO BIO
Laboratories, Carlsbad, CA, USA) according to the manufacturer’s
instructions. The purity and quality of the extracted DNA were examined
with NanoDrop Spectrophotometer (NanoDropTechnologies Inc.Wilmington,
DE, USA), and then diluted to 10 ng/μL and stored at -80 °C until use.
The abundance of bacterial and archaeal amoA was determined on a
real-time PCR detection system (Eco™, illumine, USA) using primer sets
Arch-amoAF/Arch-amoAR (Francis et al. 2005) for AOA and
amoA-1F/amoA-2R (Rotthauwe et al. 1997) for AOB. The PCR products
were purified using an AxyPrep DNA Gel Extraction Kit (Axygen, USA).
Prior to sequencing, the DNA concentration of each PCR product was
measured by the QuantiFluor™-ST blue fluorescence system (Promega, USA).
Subsequently, purified amplicons were pooled in equimolar amounts and
paired-end (PE) sequenced (2 × 300) on an Illumina MiSeq PE300 platform
at Majorbio Bio-Pharm Technology Co., Ltd., Shanghai, China
(http://www.majorbio.com).
Raw sequences were processed and analyzed using QIIME pipeline (Caporasoet al. 2010). The quality control was conducted with removing the
sequences those with a quality score < 20, containing
ambiguous nucleotides, or not matching the primer and barcode. Chimeric
sequences were checked and removed using the UCHIME algorithm (Edgaret al. 2011). Operational taxonomic units (OTUs) were identified
with UPARSE pipeline (http://drive5. com/uparse/) at 85% similarity
level (Pester et al. 2012). Taxonomy classification ofamoA gene sequences was assigned using the ARB databases for AOA
and AOB (Abell et al. 2012). An even number of sequences per
sample were selected to correct for difference in sequencing efforts
(10, 900 and 26, 190 for AOA and AOB, respectively). The raw sequences
of archaeal and bacterial amoA genes were deposited into the NCBI
Sequence Read Archive under accession numbers PRJNA637243, PRJNA637251,
respectively.