Supplemental methods
Biodegradation of dichlorodiphenyltrichloroethanes (DDTs) and
hexachlorocyclohexanes (HCHs) with plant and nutrients and their effects on the
microbial ecological kinetics
Guangdong SUN†*, Xu Zhang†, Qing Hua , Heqing Zhanga, Dayi Zhangb, Guanghe Li†*
†. State Key Laboratory of Environmental Simulation and Pollution Control, &
School of Environmental Science, Tsinghua University Beijing 100084, China
a. China Energy Conservation DADI Environmental Remediation Co., Ltd &
State Environmental Protection Engineering Center for Industrial Contaminated Sites and Groundwater Rem
ediation, Beijing 100083,China
b. Lancaster Environment Centre, Lancaster University, Lancaster, LA1 2YQ, UK
* Author for correspondence:
Guangdong SUN
School of Envrionment,
Tsinghua University
No.1 Qinghua Park, Beijing 100084, ,
P. R. China
Tel./fax: 86 10 62773232.
Email: sungd11@mails.tsinghua.edu.cn
Pyrosequencing of 16S rRNA gene amplicon libraries
The 16S rDNA was also amplified by PCR for multiplexed pyrosequencing using barcoded primers
(Supplementary Table S5). A set of primers was designed by adding a six-nucleotide barcode to the
universal forward primer Gray27F and the reverse primer Gray518R (Supplementary Table S3) for
amplification of bacteria (V1–V3 regions), 16S rDNA, and target at the hypervariable V1–V3 region. PCR
was performed with a thermal cycler (Bio-Rad, Hercules, CA, USA) under the following conditions: initial
denaturation at 94°C for 5 min; 26 cycles at 94°C for 30 s, 53°C for 30 s and 72°C for 45 s; and a final
extension at 72°C for 6 min. PCR products were detected by 1.2% agarose gel electrophoresis purified using
the TaKaRa Agarose Gel DNA Purification Kit (TaKaRa, Dalian, China) and quantified with the NanoDrop
device. A mixture of PCR products was prepared by mixing 200 ng purified 16S amplicons from each soil
sample and then pyrosequenced on the Roche 454 FLX Titanium platform at the National Human Genome
Centre of China, Shanghai, according to the manufacturer’s manual. Quality processing of 16S rRNA gene
sequences was performed in Mothur (v.1.28.0) following mainly the 454 SOP that is outlined in [1].
All statistical analyses were conducted in Mothur, JMP 8.0 (SAS Institute, Cary, NC, USA) and R
version 2.15.2 (available at http://www.R-project.org). We examined the effects of operational taxonomic
unit nucleotide similarity cutoffs on metrics such as diversity and community Bray–Curtis distance at 97%,
95% and 90%. However, as the observed patterns were similar at each cutoff, we used only 97% OTUs
(most analyses) and 90% OTUs (correlation network) for final analyses. Singleton sequences that appeared
only once in the data set were removed, and each sample was subsampled with the Mothur command
“sub.sample” to 1207 reads for 97% OTUs and 1160 reads for 90% OTUs, which was the minimum number
of sequences remaining in a single sample. The minimum number of reads per sample was higher for 90%
OTUs, because the number of singleton sequences was reduced because of inclusion within larger OTUs. To
look at the effect of different treatments on community composition, we used a de-trended correspondence
analysis [2]. The de-trended correspondence analysis transformation was performed in R using the
“decorana” command in the Vegan package with down-weighting of rare taxa. Significance of the effect of
contaminants on community structure was confirmed with a PERMANOVA on community Bray–Curtis
values using the “adonis” function in Vegan. The number of OTUs that were shared between contaminant
levels was visualized using the Mothur “venn” command.
To determine whether bacterial communities were similarly affected by different strategies, we used
Mantel tests [3] to compare bacterial community Bray–Curtis dissimilarity matrices from each treatment. In
other words, for each treatment, we compared whether the community distances for each possible pairwise
combination of samples were related for bacterial communities. We also compared the mean Bray–Curtis
dissimilarity value between all bacterial communities within each microcosm and tested for significant
differences using one-way ANOVA. Although 454 read abundance is not an exact reflection of the actual
taxonomic abundance in situ [4], standardized processing allows the detection of relative shifts between
microbial communities. The mean bacterial diversities at each contaminant level were compared using
one-way ANOVA. The composition of major bacterial classes was compared between O. violaceus at each
contaminant level using UPGMA clustering. Taxonomic abundance data were first normalized using the
“decostand” and “vegdist” commands in the Vegan package of R.
Rarefaction curves
We estimated rarefaction curves for each sample individually as well as compartment-specific rarefaction
curves using means at each sampling size for all rarefaction curves of samples belonging to that
compartment.
Bray–Curtis dendrogram
The raw OTU counts were rarefied to 1000 counts per sample employing the function “rarefy” of the R
package Vegan. Log2-transformed RA values were used to calculate a Bray–Curtis distance dissimilarity
matrix using the function “vegdist” of the R package Vegan. The dissimilarity matrix was used to generate
corresponding cluster dendrograms using the function “hclust” of the R package Vegan, specifying the
average clustering mode.
OCP extraction from soil samples
Total OCPs were extracted following the Soxhlet extraction method. All the samples were first spiked with
PCB 209 as surrogate standards prior to extraction. The 5.0 g of air-dried soil subsequently underwent
Soxhlet extraction for 24 h with hexane/dichloromethane mixture (4:1,v/v) as the extraction solvent (ASE
300; Dionex, Sunnyvale, CA, USA). To improve the extraction efficiency, anhydrous sodium sulfate (5.0 g)
and activated copper (1.0 g) were mixed with the soil samples to remove water and sulfur-containing
compounds. All extracts were concentrated with a rotary vacuum evaporator system to 1 ml, followed by
Florisil solid phase extraction treatment; the cartridge (1.0 g, 6 ml; Supelco, Bellefonte, PA, USA) of which
was filled with anhydrous sodium sulfate (2.0 g). The total OCPs were eluted with 40 ml
hexane/dichloromethane solvent (9:1, v/v), concentrated to near-dryness by rotary vacuum evaporation, and
finally reconstituted in hexane (1 ml) prior to GC analysis.
Analytical methods of OCPs
OCP analysis was performed using Agilent gas chromatography (7890B) equipped with a Ni-63 electron
capture detector and RTX-5 column (30 m × 0.25 mm i.d., film thickness: 0.25 µm). Nitrogen (99.99%
purity) was used as the carrier gas at a flow rate of 1.0 ml min–1. The injector and detector temperature were
maintained at 260°C and 300°C, respectively. The column temperature was programmed as follows: initial
temperature 100°C held for 2 min, increased to 170°C at 25°C min–1, then a ramp at 2°C min–1 to 225°C
with the temperature maintained for 2 min, to 290°C at 10°C min–1, and maintained for 8 min. One
microliter of the sample was injected in splitless mode.
Analytical methods of soil properties
Soil samples were air-dried, homogenized and sieved. The particle size distribution was measured by sieving
and sedimentation after organic matter destruction (H2O2). The fraction <2 mm was characterized for pH in
water suspension (1:2.5, w/v). The organic carbon content was determined by wet combustion. The cation
exchange capacity and exchangeable cation was measured by 1 mol l–1 ammonium acetate at pH 7.0.
Moisture was determined by placing preweighed contaminated soil samples in an oven at 105°C for 24 h.
Total organic carbon was determined according to the Walkley–Black method [5]. Total phosphorus and
total nitrogen were analyzed according to standard methods [6]. Soil respiration was measured in triplicate
with the Isermeyer method as previously described [7]. Fifty grams of sieved soil (WHC=65%) was
transferred into a beaker and placed in an airtight 1.0-l glass jar. CO2 released by basal respiration during 3
days at 25°C was trapped in 25 ml NaOH (0.05 M), which was titrated with HCl (0.05 M) after adding 5 ml
BaCl2·12H2O (0.5 M), phenolphthalein as a pH indicator.
Nucleic acid extraction and manipulation
DNA was extracted in triplicate using an MP FastDNA SPIN Kit according to the manufacturer’s
instructions. All extractions were performed immediately following soil sampling to preclude the effects of
soil storage on the microbial community. The mass of DNA in each replicate sample was quantified with a
Nandrop ND-3300 fluorospectrometer (Nanodrop Technologies, Wilmington, DE, USA) after adding
picogreen dsDNA fluorescent indicator dye. DNA and amplified products were purified using Nucleospin
Extract II PCR clean-up Gel extraction kit (Macheary-Nagel GmBH).
Nucleic acid quantification
16S rRNA and linA-like gene copies were evaluated by a Taqman or SYBR Green based real-time PCR
quantification using an iCycler iQ5 themocycler (Bio-Rad), and their primers (Supplementary Table S1)
and amplification protocols were specifically described by Suzuki et al., Cebron et al., and Huang et al.,
respectively [8, 9].
Normalization of gene copies
Copy number was normalized by Equation (1) below, where S is the normalized copy number. Cx is the
number of targeting gene copies from 1.0 g dry weight soil, and C0 is the number of copies of 16S rRNA
extracted from 1.0 g dry weight soil
𝑆=
𝐶𝑥
⁄𝐶
0
(1)
𝑥 = {𝑙𝑖𝑛𝐴 genes}
Reference
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