Detection Methanogens in Newly Settled Sediments from Xuanwu Lake in Nanjing, China

Detection methanogens in newly settled sediments from Xuanwu Lake in Nanjing, China

Dong-Lin Zhu·Cheng Sun·Huan He

D.-L Zhu·C. Sun( )·H. He

The State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment,

Nanjing University, 163 Xianlin Ave, Nanjing, Jiangsu, 210046, China.

E-mail: [email protected]

D.-L Zhu

Jiangsu Engineering consulting center, 285 North Zhongshan Road, Nanjing, Jiangsu, 21003, China

Support information

Methods and materials

Sampling and determination of methane

For each site, bamboos were embed in the sediments one day before sampling, and a static chamber with a buoy was tied the aerobic parts of the bamboos by using a 5 m-rope. The static chamber was located in the central parts of the buoy, and adhesive tape was used to ensure airproof in the sample system. The chambers included a fan to mix the air in the headspace (about 15 L) and a thin ventilating tube was used for gas sampling. Seven gas samples (30 mL) were taken in a 5-min measurement period with 60-mL polypropylene syringes equipped with three-way stopcocks. CH4 samples were analysed within 24 h of sampling with a Porapak Q column on a gas chromatograph (Agilent 6820) equipped with a flame ionization detector. CH4 flux was calculated based on the linear increase in the headspace gas concentration with time.

Methane concentrations were measured according to the methods of Jorgensen et al. (2001). Briefly, 3- mL of sediments was immediately transferred into the 25-mL Bellco anaerobic tubes containing 6 mL of 1 M NaOH solution to inhibit methanogenesis. The vials were capped immediately with butyl rubber stoppers and shaken vigorously for 1 min. Then, a 0.5-mL gas sample from the headspace was withdrawn and analyzed using above mentioned gas chromatograph. The CH4 peak was recorded on a strip-chart recorder and quantified by comparison with a methane standard curve. Each sample had three replicates.

The standard curves for quantative PCR.

1 PCR products (except for bacteria) after purification were cloned into pGEM-T® Easy vector

(Promega, WI, U.S.A.). The ligated products were transformed in competent cells of Escherichia coli,

XL1-Blue strain showing resistance to tetracycline. The transformed clones were selected on LB plates containing 100 μg/ml ampicillin and 12.5 μg/mL tetracycline. Selected clones were for the desired gene were cross checked by performing colony PCR and followed by electrophoresis on 1.5% agarose. The plasmids were selected from positive clone and the standard curves for each assay were constructed on the basis of serial 10-fold dilutions of plasmids containing cloned gene fragments of each target molecule and ranging from 1.0×102 to 1.0×109 copies per PCR. Only standard curves with regression coefficient values above 0.98 were used. All PCRs were performed in triplicate for each sample.

Reaction products were verified by 1.0% (w/v) agarose gel electrophoresis.

2 Figure S1. Quality of the amplification of bacteria, Archaea and methanogen populations in the

sediment sample from depth with 9-12 cm (Site A) by using Real-time quantitative PCR. A,

trends readed fluorescence unites in a 45 cycles of amplification; B, the melt curve of the PCR

products from 50 to 95 ºC; C, melt peak of the PCR products at 50-95 ºC; D, diagram of the

PCR products in 1.0% (w/v) agarose gel after electrophoresis. (BAC, bacterial; ARC, Archaea;

MBT, Methanobacteriales; MCC, Methanococcales; MMB, Methanomicrobiales; MSL,

Methanosarcinales; Msc, Methanosarcinaceae; Mst, Methanosaetacea).

3 Table S1. Primers used in this study (Yu Y, Lee C, Kim J, Hwang S (2005). Group-Specific Primer and Probe Sets to Detect Methanogenic Communities Using Quantitative Real-Time Polymerase Chain Reaction. Biotechnol. Bioeng., 89(6): 670-679) Primer Amplicon target group Sequence (5`→3`) Tm (oC) Name size (bp) MBT857F CGWAGGGAAGCTGTTAAGT 60.7 Methanobacteriales 343 MBT1196R TACCGTCGTCCACTCCTT 63.2 MCC495F TAAGGGCTGGGCAAGT 60.4 Methanococcales 337 MCC832R CACCTAGTYCGCARAGTTTA 61.6 MMB282F ATCGRTACGGGTTGTGGG 63.8 Methanomicrobiales 506 MMB832R CACCTAACGCRCATHGTTTAC 61.5 MSL812F GTAAACGATRYTCGCTAGGT 61.3 Methanosarcinales 354 MSL1159R GGTCCCCACAGWGTACC 62.3 Msc380F GAAACCGYGATAAGGGGA 61.2 Methanosarcinaceae 408 Msc828R TAGCGARCATCGTTTACG 59.9