Microbial Biomass Concentration and DNA Extraction

The microbial biomass of each sample was concentrated by filtration using the Millipore vacuum/pressure pump (Millipore Corporation, Bedford, Mass.). Each 250-mL sample was mixed with 1/4 volume of saturated sodium chloride solution and incubated at 70°C for 2 min before filtration (Ren et al., 2011 (link)). A 0.22-μm filter (Millipore, USA) was used to collect microbial biomass following an 8-μm filter (Bandao, China). All filters of one sample were collected and stored in a sterilized 10-mL centrifuge tube at −20°C for DNA extraction. The genomic DNA was extracted from the filters using a bead-beating protocol as described previously (Zhang et al., 2007 (link)). The AxyPrep PCR cleanup kit was used to purify genomic DNA, which was stored at −20°C until 16S rRNA gene amplification.