Evidence for extensive anaerobic dechlorination and transformation of the pesticide chlordecone (C10Cl10O) by indigenous microbes in microcosms from Guadeloupe soil

The historic use of chlordecone (C10Cl10O) as a pesticide to control banana weevil infestations has resulted in pollution of large land areas in the French West Indies. Although currently banned, chlordecone persists because it adsorbs strongly to soil and its complex bis-homocubane structure is stable, particularly under aerobic conditions. Abiotic chemical transformation catalyzed by reduced vitamin B12 has been shown to break down chlordecone by opening the cage structure to produce C9 polychloroindenes. More recently these C9 polychloroindenes were also observed as products of anaerobic microbiological transformation. To investigate the anaerobic biotransformation of chlordecone by microbes native to the French West Indies, microcosms were constructed anaerobically from chlordecone impacted Guadeloupe soil and sludge to mimic natural attenuation and eletron donor-stimulated reductive dechlorination. Original microcosms and transfers were incubated over a period of 8 years, during which they were repeatedly amended with chlordecone and electron donor (ethanol and acetone). Using LC-MS, chlordecone and degradation products were detected in all the biologically active microcosms. Observed products included monohydro-, dihydro- and trihydrochlordecone derivatives (C10Cl10-nO2Hn; n = 1,2,3), as well as “open cage” C9 polychloroindene compounds (C9Cl5-nH3+n n = 0,1,2) and C10 carboxylated polychloroindene derivatives (C10Cl4-nO2H4+n, n = 0–3). Products with as many as 9 chlorine atoms removed were detected. These products were not observed in sterile (poisoned) microcosms. Chlordecone concentrations decreased in active microcosms as concentrations of products increased, indicating that anaerobic dechlorination processes have occurred. The data enabled a crude estimation of partitioning coefficients between soil and water, showing that carboxylated intermediates sorb poorly and as a consequence may be flushed away, while polychlorinated indenes sorb strongly to soil. Microbial community analysis in microcosms revealed enrichment of anaerobic fermenting and acetogenic microbes possibly involved in anaerobic chlordecone biotransformation. It thus should be possible to stimuilate anaerobic dechlorination through donor amendment to contaminated soils, particularly as some metabolites (in particular pentachloroindene) were already detected in field samples as a result of intrinsic processes. Extensive dechlorination in the microcosms, with evidence for up to 9 Cl atoms removed from the parent molecule is game-changing, giving hope to the possibility of using bioremediation to reduce the impact of CLD contamination.


A) Collection of Field Samples
All agricultural soil samples were collected from 0-20 cm depth. In 2010, soil, water and sludge samples were collected separately in polypropylene sampling bottles, while in 2018, a mix of soil and water was placed into 1-liter glass sampling jars that were filled to the top and sealed. All samples were shipped from Guadeloupe to University of Toronto for analysis where they were stored at 4 o C until use.

B) Recipe for Artificial Groundwater Used in Microcosm Setup
From Middeldorp et al. (1998).
For 1 liter of water, the following compounds were added: The solution was made with distilled deionized water. It was autoclaved and then purged with N2/CO2 (80% /20%) for 1 hour. After, the artificial groundwater was moved into the glovebox. In the glovebox 10 mL of 100x sterile vitamins solution was added (recipe in Edwards and Grbić-Galić, 1994). The pH was measured and adjusted to pH 7.

A) GC-FID Sample Preparation and Analysis
Microcosms were sampled using glass syringes and a 1 ml sample was added to a 10 ml headspace autosampler vials (Agilent) containing 5 ml of acidified deionized water (pH 2). The vials were then sealed with Teflon-coated septa and aluminum crimp caps (Chromatographic Specialties) for automated headspace injection onto the CG. Methane, ethene, ethane, and chlorinated ethene measurements were carried out as headspace analysis using an Agilent 7890A gas chromatograph (GC) equipped with a flame ionization detector (FID), a G1888 headspace autosampler and a J&W GS-Q column (30 m x 0.53 mm) (Agilent, Santa Clara, CA, USA). Helium was the carrier gas (11 ml/min) and the oven program was as follows: Hold at 35°C for 1.5 min, increase to 100°C at the rate of 15°C/min, then increase to 185°C at the rate of 5°C/min, hold 10 min, then increase to 200 °C at the rate of 20°C/min, hold 10 min (total runtime 43.6 min). The GC had a packed inlet and a 3 ml sample loop. Headspace operating temperatures for oven, loop and transfer line were 70, 80 and 90 o C respectively, while vial equilibration time, pressurization time, loop fill time, loop equilibration time and injection time were 40, 0, 0.2, 0 and 3 min respectively. During equilibration in oven, the samples were shaken at low speed. Data from the GC was integrated using ChemStation (Agilent). Calibration standards were prepared in the concentration range 0.2 to 2 mg/l (liq) for methane and ethene, and 1 to 20 mg/l for chlorinated ethenes.

B) IC Sample Preparation and Analysis
To measure chloride, nitrite, nitrate, sulfate, phosphate and acetate concentrations, samples (1 ml) were filtered through 0.2 μm nylon filters (Fisher Scientific). Analysis were performed using a Dionex ICS 2100 ion chromatograph with a Dionex IonPac AS18 analytical column (4x250 mm) and an ASRS 500 suppressor (Thermo Fisher Scientific). The samples were run isocratically at 23 mM KOH, 57 mA current and with a flow rate of 1 ml/min. Data from the IC was integrated using Chromelion (Thermo Fisher Scientific). Standards were prepared in the range 0.005 to 0.5 mM.

C) pH Measurements
One ml liquid samples were taken for "in-syringe" pH analysis using an Oakton pH spear (Oakton Instruments, Vernon Hills, USA). If needed, pH was adjusted with Na-bicarbonate or HCl to pH 7.

Details of the different sample preparation methods
Sample preparation method 1 (water phase with small amount of soil, liq/liq extraction): 1) Let soil settle in bottles overnight 2) Sample 2 mL liquid from microcosms 3) Extract with 15/85 % acetone/hexane, 2 cycles of extraction, each w/ 5mL of acetone/hexane 4) Filter the solvent phase through hydrophobic filter into glass vial 5) Evaporate filtrate to dryness and re-dissolve in 2mL of MeOH Sample preparation method 2 (water phase): 1) Let soil settle in bottles overnight 2) Sample 1 mL liquid from microcosms 3) Filter sample into new glass vial through 0.2 μm PTFE syringe filter Sample preparation method 3 (water phase): 1) Let soil settle in bottles overnight 2) Sample 0.75mL liquid from microcosms, avoiding getting any soil into the sample, and transfer to glass vial 3) Centrifuge @ 3000rpm for 5min 4) Transfer 0.5mL clear liquid from centrifuged samples into glass vials with 0.5mL MeOH, mix 5) Filter sample into new glass vial through 0.2 μm PTFE syringe filter

Sample preparation method 4 (samples with soil):
1) Shake bottle and sample 1mL slurry from microcosm 2) Add to glass vial containing 1mL MeOH, vortex, shake gently for 10min and let sit for about 30 min (or overnight) 3) Centrifuge @ 3000rpm for 5min 4) Filter sample into new glass vial through 0.2 μm PTFE syringe filter Sample preparation method 5 (extraction of field samples, 5 ml slurry): 1) Shake bottle and sample 5mL slurry from sampling jar into a 15 ml glass centrifuge tube 2) Add 5 ml of a 15% acetone and 85% hexane mixture to the centrifuge tube 3) Shake gently for 10 min, let sample sit for 1 hour, shake for 1 min 4) Centrifuge @ 3000 rpm for 5min 5) Transfer the solvent phase into new glass vial 6) Filter sample into new glass vial through 0.2 μm PTFE syringe filter 7) Evaporate to dryness 8) Re-dissolve in 1 ml methanol Sample preparation method 6 (extraction of field samples, 20 ml slurry): 1) Shake bottle and sample 5mL slurry from sampling jar into a 15 ml glass centrifuge tube (prepare 4 tubes with 5 ml sample for each field sample) 2) Add 5 ml of a 15% acetone and 85% hexane mixture to the centrifuge tube 3) Shake gently for 10min and let sample sit for 30 min 4) Centrifuge @ 3000 rpm for 5min 5) Transfer the solvent phase into new glass vial (combine solvent from the 4 tubes into one vial) 6) Repeat steps 2 to 5 one more time 7) Evaporate to dryness 8) Re-dissolve in 0.5 ml MeOH 9) Filter sample into new glass vial using 0.2 μm PTFE syringe filter

E) DNA Extraction, Amplicon Sequencing and Quantitative Polymerase Chain Reaction (qPCR) Analysis
Samples for DNA extraction and subsequent microbial community analysis were taken at different time points from the 6 transfers (GT5, GT20, GT33, GT4, GT15, GT3) and one TCE amended control (GT2). Slurry samples (1 mL) were collected, centrifuged at 10 000 rpm for 20 min, and cell pellets were frozen at -80 o C for future DNA extraction. DNA was extracted using the DNeasy PowerSoil Kit (Qiagen, Hilden, Germany) according to manufacturer's protocol with some modifications (see below). DNA concentrations were verified by a NanoDrop1000 Spectrophotometer (Thermo Fisher Scientific) and Qubit Fluorometric Quantitation (Thermo Fisher Scientific), and the extracts were stored at -80 o C.
Microbial community composition of the samples was assessed by small subunit (SSU) rRNA gene fragment sequencing and Quantitative Polymerase Chain Reaction (qPCR) analysis. Samples from earlier sampling times (after 29 and 39 months) were sequenced using Pyrotag 454 sequencing and samples from later timepoints (76 and 79 months) were sequenced using Illumina MiSeq sequencing.

Pyrotag 454 sequencing
Samples were sequenced at Genome Quebec Innovation Centre using the Roche GS FLX Titanium technology (Roche Diagnostics Corporation, Indianapolis, IN). Extracted DNA samples were amplified by PCR using the universal primer set, 926f and 1392r (926f: 5'-AAACTYAAAKGAATTGACGG-3'; 1392r: 5'-ACGGGCGGTGTGTRC-3'), targeting the V6-V8 variable region of the 16SrRNA gene from bacteria and archaea, as well as the 18S rRNA gene in eukarya (Engelbrektson et al. 2010 . The forward and reverse primers included adaptors, and the reverse primer also included 10bp multiplex identifiers (MID) for distinguishing multiple samples pooled within one sequencing region. The PCR products were verified on a 2% agarose gel and replicates were combined and purified using GeneJETTM PCR Purification Kit (Fermentas, Burlington, ON), according to the manufacturer's instructions. The concentrations of PCR products were determined using a NanoDrop ND-1000 Spectrophotometer at a wavelength of 260 nm. The concentrations and qualities of the final PCR products were also evaluated by running them on 2% agarose gels and comparing band intensities to those from a serial dilution of ladders with known DNA concentrations. The purified PCR products were sent to Genome Quebec Innovation Centre, where they were checked for quality again, pooled and subject to unidirectional sequencing (i.e. Lib-l chemistry) of the 16S gene libraries, using the Roche GS FLX Titanium technology (Roche Diagnostics Corporation, Indianapolis, IN).

Processing of amplicon sequencing
Sequence data from both sequencing technologies was processed in QIIME2 v 2019.10 ( Bolyen, E., et al. 2019). Primers were removed using the cutadapt plug-in QIIME2. Amplicon sequence variants (ASVs) were identified using the QIIME2 DADA2 plugin with the following settings; forward reads were trimmed to 260nt and reverse reads to 220, maximum expected errors both for forward and reverse reads were set to 3. Taxonomy was assigned to the ASVs using the feature-classifier plugin and machine-learning-based classification (the classify-sklearn option) using a SILVA v.132 classifier trained on the region amplified by the primers used here. The 454 sequence data was demultiplexed and primers were removed in Geneious v10. The resulting reads were processed by DADA2 V1.1 in R with sequences trimmed to 400 nt, maximum errors set to 2 and homopolymer_gap_penalty=-1, band_size=32 to generate ASV (Callahan, B. J., et al. 2016). Taxomony was assigned to the ASVs using the 'assignTaxonomy' function in the DADA2 package with the SILVA v.132 database.
The ASVs obtained from both sequencing chemistries were imported into Geneious v.10 and aligned using MAFFT v7.450. Positions containing > 99% gaps were removed from the alignment, and 68bp at the 5'-end and 12bp at the 3'-end of the alignment were trimmed off, resulting in a 413bp alignment of 7966 ASVs. The alignment was used to produce a phylogenetic tree using FastTree v. 2.1.1.11. The ASV tables from both datasets were imported to Phyloseq v. 1.26 (McMurdie, P. J. and S. Holmes 2013) in R together with the phylogenetic tree. The ASVs were combined into operational taxonomic units (OTUs) using the tip-glom function in phyloseq with h=0.03. This function agglomerates all tips of the tree separated by a distance smaller than h into one taxon or OTU. This gave 4935 combined OTUs. The dataset was subsampled without replacement to an equal number of 4000 reads pr sample and a bar chart of the 100 top OTUs was drawn using the Fantaxic R-package (https://rdrr.io/github/gmteunisse/Fantaxtic/).

qPCR analysis
The abundance of total bacterial 16S rRNA genes in the microcosms was estimated by qPCR using a CFX96TM real-time PCR detection system, with a C1000 thermocycler (Bio-Rad Laboratories Inc., Hercules, CA, USA). DNA extracts were analyzed using the general bacterial 16S rRNA primers 055f (5'-ATGGCTGTCGTCAGCT-3') and 1392r (5'-ACGGGCGGTGTGTAC-3') (Ferris et al. 1996). The 20 μl qPCR reactions were prepared in a PCR cabinet (ESCO Technologies, Gatboro, PA) and were made up by 10 μl of SsoFastTM EvaGreen® SuperMix (Bio-Rad Laboratories Inc., USA), 0.5 μl of each forward and reverse primers (10uM stock, making final concentration of 250 nM for both primers), 7 μl of UV treated UltraPure Distilled water (Invitrogen, Grand Island, NY, USA), and 2 μl of DNA extract diluted 10x. The qPCR cycle was as follows: 98°C for 2 min, 40 cycles of 98°C for 5 seconds and 55°C for 10 seconds, followed by an increase from 65°C to 95°C at 0.5°C increments over 10 seconds. Calibration curves for qPCR were prepared from serial dilutions of target-containing plasmids between 10 1 and 10 8 gene copies/ml. The general bacteria gene copy number detection limit was 1.1 E05 copies per ml.

Modifications to the DNeasy PowerSoil Kit manufacturer's protocol
Link to Dneasy PowerSoil kit Protocol: https://www.qiagen.com/ca/resources/resourcedetail?id=91cf8513-a8ec-4f45-921e-8938c3a5490c&lang=en Modifications to protocol: Step 1: Cell pellet was added to the tube (instead of soil sample) Step 9: All of the supernatant was transferred to the collection tube (not only 600 μl) Step 12: All of the supernatant was transferred to the collection tube (not only 750 μl) Step 13: Keep the ratio of sample to C4 solution at 750:1200 (sample:C4) Step 19: Added 50 μl H2O instead of 100 μl C6  Figure S4: TCE and its dechlorinated metabolites in a CLD+TCE amended microcosm during the first 2 years of monitoring (microcosm G15). The two other triplicates (G13 and G14) behaved similarly. TCE continued degrading in G15 and after transferring in GT15, but GT3 which was a transfer from G14 and G15, never degraded TCE ( Figure S2 and Table S5)