Introduction

Pentachlorophenol (PCP) is a highly toxic and persistent chemical that is classified as a priority pollutant by the US Environment Protection Agency (Keith and Telliard 1979). Since the 1980s, PCP has been widely used as a pesticide in Chinese paddy fields, which has negatively influenced the agricultural soil ecosystems (Augustijn-Beckers et al. 1994). Although banned as an insecticide in China by 1997, PCP residue in soils and groundwater still remains a widespread environmental concern because of its high toxicity and molecular stability (Augustijn-Beckers et al. 1994). As a result, the transformation of PCP in the environment has been the focus of attention for a considerable time. It is well known that PCP can be biodegraded under both aerobic and anaerobic conditions in paddy soils (Li et al. 2013). In the flooded paddy soils, PCP was observed to dechlorinate to less chlorinated phenols (Kuwatsuka and Igarashi 1975; Yoshida et al. 2007). According to those reports, PCP is expected to be dechlorinated by microorganisms under anaerobic conditions like flooded paddy soils.

Anaerobic bacteria capable of dechlorination were isolated from enrichment cultures, and exhibited excellent tolerance and rapidly dechlorinated PCP. For example, Desulfitobacterium frappieri PCP-1, isolated from a methanogenic consortium, can not only dechlorinate high concentrations of PCP, but also dechlorinate several different chlorophenols at ortho, meta, and para positions (Bouchard et al. 1996). Due to limitation of the culture-dependent methods, only a small proportion of the dechlorinating microbiota has been successfully isolated and cultivated from dechlorination cultures. Therefore, exploring potential functional diversity of the microbial community that is responsible for dechlorination of PCP is important part of bioremediation research. Anaerobic dechlorination of PCP forming lesser chlorinated phenols has been observed in sediments and soils environment (Kennes et al. 1996; Xu et al. 2015; Kranzioch-Seipel et al. 2016). Yoshida et al. (2007) reported that an anaerobic microbial community could dechlorinate PCP to phenol in paddy soils supplemented with lactate, thereby leading to the physiological characterization of novel bacteria in the Firmicutes phylum. In the subsequent studies about this enriched microbial community, Zhang and Katayama (2012) and Zhang et al. (2014) founded that the humic substances in the soils were essential factor for sustaining the dechlorination activity and functioned as redox mediator in PCP dechlorinating cultures. In the iron rich paddy soil, the generated Fe(II) species by iron-reducing microorganisms can act as reactive reductants in the biodegradation of PCP, which the possible dechlorination mechanism by microbial community was coupled with the biochemical electron transfer processes between iron redox cycling and reductive dechlorination (Chen et al. 2012; Xu et al. 2015).

Essentially, the anaerobic dechlorination of PCP is a redox reaction involving microbial respiration that dechlorinates PCP by using low molecular weight organic acids (such as acetate, lactate, and glucose) as electron donors in soils (Chang et al. 1996; Zhang et al. 2010; Payne et al. 2011). Various ionic species in soils such as Fe(III), NO3 −, and SO4 2− act as competitive electron acceptors for microorganisms with dechlorinating respiration, which influences the abundance and activity of dechlorinating bacteria (Adriaens et al. 1996; Kotik et al. 2013). During dechlorinated respiration, the soils of different soil types with the different contents of low molecular weight organic acids and ionic species impacted the microbial community structure, further affecting the reduction of PCP (Kuwatsuka and Igarashi 1975; Chen et al. 2014). In paddy soils, acetate is the most important metabolite of the anaerobic food chain (Hori et al. 2010) and dechlorination of PCP has been observed previously (Yoshida et al. 2007). However, there is poor information about the microbial community associated with the acetotrophic PCP dechlorination in iron rich paddy soil.

In this study, the microbial community responsible for PCP dechlorination was enriched through the serial transferred incubations in medium containing acetate, PCP in iron-rich soil. To further explore the succession of microbial communities at different intervals and the main metabolic processes of microorganisms during the PCP dechlorination, the members of this bacterial consortium were further characterized with high throughput sequencing of 16S rRNA. In addition, specific detection of dechlorinating bacteria, such as Dehalobacter spp. and Desulfitobacterium spp. were conducted by quantitative PCR (qPCR) using a set of the genus-specific primers.

Materials and methods

Soil sampling and chemicals

The soil sample was collected 15 cm beneath the surface of paddy soil in Sijiu Village of Jiangmen City, China (22o12′50.90″N, 112o53′4.03″E), in August 2012. After collection, the samples were quickly extruded into glass tubes, stoppered, and immediately transported back to the laboratory and stored in an anaerobic chamber to maintain anoxic conditions for one day before the experiments. No chlorinated phenols were detected in soil and our previous studies have found the chlorinated compounds can be degraded by the microbial communities in adjacent soil (Tong et al. 2014, 2015). The basic physicochemical properties of the soil were analyzed with a previously described method (Pansu and Gautheyrou 2007), and the results were as follows: 17.5% Al2O3, 63.6% SiO2, 20.3 g kg−1 total organic carbon, 20.4 g kg−1 total Fe, and 44% moisture content. The contents (weight/weight) of clay, sand, and silt in the soil were 33, 38, and 29%, therefore, the soil can be classified as clay loam, based on the USDA soil textural triangle standard (Staff 2014). Piperazine-1,4-bis (2-ethanesulfonic acid) (PIPES, 99%) and sodium acetate (99%) were purchased from Sigma Aldrich (USA). All other chemicals of analytical grade were obtained from Guangzhou Chemical Co. (Guangzhou, China). Deionized water (18.2 MΩ cm) was prepared using an ultrapure water system (EasyPure II RF/UV, ThermoScientific, USA) and used in all experiments.

Incubation experiments

To activate the soil microorganisms and reduce a small amount of electron acceptors such as available sulfate and nitrate, the soil was pre-incubated with 65% water content in the airtight bottles at 25 °C for several days before use (Yoshida et al. 2007; Hori et al. 2010). For the preparation of the dechlorination culture, ~10 g soils (wet weight) were introduced into 100 mL serum bottles containing 25 mL medium and were flushed with nitrogen gas (0.1 MPa, 99.99%) for 30 min, and then sealed with butyl rubber stoppers and aluminum crimp seals. The vitamin and mineral solution (Holliger et al. 1998) were added to the medium through a 0.22 μm membrane filter. The medium consisted of (per liter) 30 mM (9.1 g L−1) PIPES buffer (pH 7.0), 1 mL vitamin solution, and 1 mL mineral solution. Sterile, anoxic solutions of PCP and acetate were added to final concentrations of 37.6 μM (10 mg L−1) and 10 mM (600 mg L−1) using sterilized syringe. Each bottle was wrapped twice with aluminum foil to prevent the effect of light on PCP degradation. And this process (the soil transferred from pre-incubated culture) was identified as 1st cycle. After 13 days (upon the depletion of PCP) of incubation, 2.5 mL of the homogenized slurry was transferred to 22.5 mL of a new medium, which contained 30 mM PIPES, 37.6 μM acetate and 10 mM PCP. This transfer was equivalent to 2rd cycle. The serially transferred was performed by using 10% (vol/vol) inocula when PCP was completely removed. Acetate and PCP were added to each cycle using sterilized syringe. The experiments were conducted in duplicate. After flushed with nitrogen, the preparation of all microcosms and serially transferred were done in an anoxic glovebox (100% N2 atmosphere) in order to maintain an anaerobic condition for PCP dechlorination, and the experimental reactors were incubated at 25 °C in an anaerobic chamber without shaking (Shellab, Sheldon Manufacturing Inc., Cornelius, OR).

Chemical analytical methods

During the incubation period, triplicate bottles were taken out for chemical analysis at regular intervals with each chemical determination of each bottle performed without technical replicate. To determine acetate, an aliquot of sample was filtered using a 0.45-μm syringe filter. The concentration of acetate was determined in the filtrate by a Dionex ICS-90 ion chromatograph with an ion column (Ionpac AS14A 4 × 250 mm). Samples were eluted with a 1.8 mM carbonate and 1.7 mM bicarbonate eluent at a flow rate of 2 mL min−1. Dilute sulfuric acid (13.6 mM) was used as a regenerant (Coby et al. 2011). The HCl extractable Fe(II) in the reaction suspension was extracted using 0.5 M HCl for 1.5 h, and analyzed using the 1,10-phenanthroline colorimetric method at 510 nm (Fredrickson and Gorby 1996). To determine PCP, 2 mL soil suspension from the bottle was extracted with 2 mL of water/ethanol (1:1 in volume) by shaking on a horizontal shaker at 180 rpm for 1 h (Khodadoust et al. 1999). The water–ethanol–soil mixture was filtered through the 0.45-mm filter. The filtrate was collected for HPLC analysis of PCP concentration by high performance liquid chromatography (HPLC) using a Waters Alliance 1527-2487 HPLC system fitted with a Symmetry C18 column (5 μm, 4.6 × 250 mm, Waters, USA) (Tong et al. 2014). The PCP transformation intermediates in the suspension were extracted with hexane and identified with Gas Chromatography/Mass Spectrometry (GC/MS) on a Thermo Trace-DSQ-2000 with electron ionization and an Agilent silicon capillary column (0.25 mm × 30 m) (Tong et al. 2014).

DNA extraction, 16S rRNA gene amplification, qPCR and high throughput sequencing

The sample suspensions were centrifuged and ~0.25 g soil was collected. The DNA in the soil was extracted using a PowerSoil™ DNA isolation kit (MO BIO Laboratories, Inc., Carlsbad, CA, USA) according to the manufacturer’s instructions. The DNA was quantified with a Qubit 2.0 fluorometer DNA (Invitrogen, NY, USA). The abundance of dechlorinating bacteria, such as Desulfitobacterium spp. and Dehalobacter spp. in the suspension cultures were determined by quantitative polymerase chain reaction (qPCR) on a MyiQ™ 2 Optics Module (BIO-RAD, USA), and the procedures were performed according to that of Tong et al. (2014). The total genomic DNA extracts were submitted for high-throughput amplicon sequencing at Magigen Biotechnology (Shenzhen, China) following the protocol described elsewhere (Caporaso et al. 2011, 2012). PCR amplification of 16S rRNA gene fragments was performed using primers F515 (5′-GTGCCAGCMGCCGCGGTAA-3′) and R806 (5′-GGACTACVSGGGTATCTAAT-3′), with a sample-specific 12-bp barcode added to the reverse primer (Caporaso et al. 2011). Each DNA sample was amplified in 30 μL reaction mixtures in triplicate using the following PCR program: at 95 °C for 3 min; 35 cycles at 94 °C for 30 s, annealing at 50 °C for 1 min, and extension at 72 °C for 1 min; and a final extension of 10 min at 72 °C. Illumina specific fusion primers were used to amplify the V3–V4 region of the 16S ribosomal RNA (rRNA) gene, and then unique barcodes were added into samples in each well to enable pooling and sequencing. The bioinformatics analysis was processed using Mothur and QIIME (Schloss et al. 2009; Caporaso et al. 2011). The chimeric and low quality sequences were identified and removed, whereas the 12-bp barcode was examined in order to assign sequences to individual samples. Operational taxonomic units (OTUs) were identified at the 97% sequence similarity level using UCLUST (Edgar 2010), and a representative sequence from each phylotype was selected using PyNAST (DeSantis et al. 2006). The taxonomic classification of each phylotype was determined using the Ribosomal Database Project (RDP) at the 80% threshold. Relative abundance (%) of individual taxa within each community was estimated by comparing the number of sequences assigned to a specific taxon versus the number of total sequences obtained for that sample.

Results and discussion

PCP dechlorination culture

PCP and acetate were consumed simultaneously in the PCP-dechlorinating culture at a circumneutral pH in paddy soil (Fig. 1). At the beginning and the end of the incubation of each cycle, pH values did not changed markedly (data not shown), and similar relative stability of pH has been also observed in other PCP dehclorination cultures (Yoshida et al. 2007; Chen et al. 2016). Acetate concentration was measured in the first four cycles (C1–C4, C1 was equivalent to 1st cycle), and some acetate persisted throughout the experiments durations, whereas PCP was transformed by day 10–13. The paddy soil used in this study is iron-rich soil with a high content of total iron (20.4 g kg−1). Adsorbed Fe(II) species are critical to accelerating the reductive process of organochlorine pollutants (Chen et al. 2012), and 0.5 M HCl-extractable Fe(II) has been shown to be effective in extracting produced Fe(II). The concentrations of 0.5 M HCl-extractable Fe(II) were 12.5 and 10.7 μM in the 1st and 2nd cycle, respectively. These results indicate that the culture-induced dechlorination occurred coupled with acetate metabolism and iron reduction. This was consistent with the predominance of phylotypes related to known Firmicutes and Proteobacteria taxa in the microbial community analysis data (Fig. 2). During the microbial degradation of PCP, several intermediates were detected by GC–MS, including 2,3,4,5-tetrachlorophenol (2,3,4,5-TeCP), 3,4,5-trichlorophenol (3,4,5-TCP), and 4-chlorophenol (4-CP), in line with other reports with adjacent sampling points (Tong et al. 2014). The dechlorination pathway of this study is also consistent with the microbial reductive of PCP in river sediments by Kranzioch-Seipel et al. (2016). The dechlorination pathway of the enriched microcosm indicated the preferable dechlorination in ortho-position following to meta-positions.

Fig. 1
figure 1

The PCP and acetate concentration in the cycling cultures. The arrows showed the subsequent additions of PCP and acetate

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Fig. 2
figure 2

Relative abundances (>1%) of the dominant microbial phyla in different cycles revealed by 16S rRNA high throughput sequencing. T0 represents the original soil sample; C1 represents 1st cycle

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A total of eleven cycles were carried out with fresh PCP (37.6 μM) and acetate (10 mM) added to the cultures at each time interval (Fig. 1). As each cycle progressed, the rate of PCP dechlorination increased and the dechlorination cycle time decreased. The maximum dechlorination rate was obtained at the 11th cycle, in which 8.6 mg L−1 of PCP was completely degraded within 5 days. This could be the result of the lag time necessary for microorganism activation (Lendvay et al. 2003). The microbial community structure and PCP dechlorination rate became relatively stable after 8th transfer (Figs. 1, 4). When the certain time for microbial activation, both abiotic and biotic reactions contributed to PCP removal in the cultures. In the first cycle, the indigenous organic matter can act as an electron donor for microorganism respiration. Added electron donors, such as acetate, formate, pyruvate, and lactate, can further support and stimulate the biotic dechlorination processes (Brahushi et al. 2004). Acetate is a favorable electron donor for the respiration of a wide range of microorganisms in the processes of contaminant transformation (Chang et al. 1996). More specifically, the dechlorinating and iron-reducing bacteria can utilize acetate as the electron donor to accelerate the reductive dechlorination of PCP (He et al. 2002; Chen et al. 2012). In the previous report, acetate was absolutely required for PCB-dechlorinating bacteria growth and dechlorinating activity (Cutter et al. 2001), and transformed to carbon dioxide and methane (Kittelmann and Friedrich 2008). In the present culture, acetate served as the electron donor for dehalorespiring bacteria, while acetate oxidation coupled to PCP reduction was a highly exergonic process for dehalorespiring bacterial growth (Kittelmann and Friedrich 2008), e.g. as presented in Eq. (1)

$${\text{Acetate}}^{ - } + {\text{ H}}^{ + } + {\text{ 4PCP }} + {\text{ 2H}}_{ 2} {\text{O }} \to {\text{ 2CO}}_{ 2} + {\text{ 4TeCP }} + {\text{ 4Cl}}^{ - } + {\text{ 4H}}^{ + } .$$
(1)

Microbial community diversity and composition during incubation

After adding PCP and acetate during the reaction period, the cultured microbial community acclimated and changed over time. Approximately 578,791 quality sequences for the 12 analyzed samples were generated with the Illumina high throughput sequencing, with sequences frequencies for individual samples ranging from 11,775 to 39,116 (Table 1). In total, 11,292 operational taxonomic units (OTUs) were identified in the complete data set, with an average of 3432 OTUs per sample. Over 99% of the OTUs were assigned to a taxonomic group (phylum), and over 80% were identified at the order level.

Table 1 Summary of the 16S rRNA sequences, operational taxonomic units (OTUs), and microbial diversity indices for all cycles
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The dominant phyla in the original soil sample without any amendment (T0) were the following: Firmicutes, Nitrospirae, Proteobacteria, Crenarchaeota, Chloroflexi, Bacteroidetes, and Acidobacteria. A relative abundance >4% for each phylum accounted for >85% of the total sequences (Fig. 2; Table S1), with dominant taxa being roughly consistent with those reported in the previous study on soil bacterial community compositions (Li et al. 2016). The dominant phyla in the treatment without PCP were similar with the original soil sample after 1st cycle, and the date was provided in supporting information (Figure S1). However, during the PCP and acetate treatment, the dominant phyla changed to Euryarchaeota, Bacteroidetes, Firmicutes, Proteobacteria, Chloroflexi, and WWE1, which were largely different with that in T0. In the previous study, the microbial taxonomic composition in the paddy soil amended with PCP after 40 days exhibited significant differences with the original soil and the treatment without PCP (Chen et al. 2016). The relative abundance of the different phyla varied considerably. During the 1st–4th cycle of PCP dechlorination, the relative abundance of Firmicutes decreased from 36.0 to 11.6%, and then increased to 31.5% during the 5th–11th cycle, whereas that of Proteobacteria increased from 9.9 to 29.9% during the 1st–4th cycle, and then markedly decreased to 7.9% after the 6th cycle. The relative abundance of Chloroflexi increased to 16.6% during the first two cycle, and then maintained an average value of 4.3% during the 7th–11th cycle. The relative abundance of Bacteroidetes and WWE1 increased from 4.7 to 14.1%, and 0.1 to 12.3% during the 1st–11th cycle, respectively. For the archaea, the majority of sequences were affiliated with Euryarchaeota, which the relative abundance increased markedly from 1.5 to 27.2% at end of 7th cycle, and then maintained an average value of 12.0% during the 8th–11th cycle.

At the genus level, the composition of the microbial community was obviously different and displayed a distinct succession between the initial and following cycles (Fig. 3; Table S2). The dominant genera in the original soil sample were Clostridium, GOUTA19, unclassified Nitrospirales, unclassified Anaerolineae, Anaeromyxobacter, Bacillus, unclassified Bacteroidales, unclassified Crenarchaeota, and Methanobacterium (relative abundance >1%). In the treatment without PCP after 1st cycle, besides the dominant genera mentioned in the original soil, Anaeromusa was also enriched in the culture (Figure S2). However, the relative abundance of Anaeromusa (3.2%) in the treatment without PCP was lower than that in the treatment with PCP (9.3%) indicated Anaeromusa was likely to play an important role in PCP dechlorination coupled with acetate oxidation. With the amendment of PCP and acetate, the relative abundances of Methanosarcina and Anaeromusa evidently increased after the 2nd cycle. The relative abundance of Methanosarcina achieved an average 7.7% during the 2nd–7th cycle, and then decreased to an average value of 2.5% after the 8th cycle. The relative abundance of Anaeromusa dramatically decreased to 3.7% at the 4th cycle, and then steadily increased to 21.2% at the 11th cycle. The relative abundance of Zoogloea dramatically increased to 21.6% during 2nd–4th cycle, but decreased to approximately 1% at the 11th cycle. Syntrophobotulus slightly increased over throughout the whole process, and the relative abundance maintained at an average value of 1.6% during the 8th–11th cycle. The relative abundances of Treponema, W22, and unclassified SHA-116, however, increased continuously to 13.7, 5.0, and 4.6%, respectively, at the 11th cycle. The changes in these cycle communities were analyzed using a principal component analysis (PCA), and the results are presented in Fig. 4. The results show that C6–C11 group relatively close together, which demonstrates that microbial communities stability increased as cycles increased (Li et al. 2016). In the enrichment culture, PCP dechlorinating microbial community structure by serial transfers from a paddy soil become stable in dechlorination in different cultures (Yoshida et al. 2007; Zhang and Katayama 2012). To further explore dechlorinating bacteria, such as Desulfitobacterium spp. and Dehalobacter spp. in the cycles of PCP incubation, 16S rRNA gene copy numbers of the dechlorinating bacteria were measured by qPCR. However, the copy numbers of these dechlorinating bacteria were very low, even not observed (Figure S3). To date, only few PCP respiring isolates are known to use acetate as a direct electron donor (Field and Sierra-Alvarez 2008). The dominant genera in the enrichment culture were seldom associated with PCP dechlorination coupled with acetate oxidation. In our study, the extent of direct acetotrophic PCP dechlorination by soil microorganisms remains elusive.

Fig. 3
figure 3

The dominant microbial genera based on their relative abundance (>1%) in different cycles (z-score normalized across all taxa, indicated by the color key). T0 represents the original soil sample; C1 represents 1st cycle

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Fig. 4
figure 4

Principal component analysis of microbial communities obtained from different cycles

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Methanosarcina has been reported as a reductive dechlorinating microorganism under anaerobic conditions (Wang et al. 2000). The isolation of Methanosarcina from methanogenic and sulfate-reducing consortium was able to dechlorinate high concentrations of tetrachloroethylene, 2,4-dichlorophenol and 2,4,6-trichlorophenol (Wang et al. 2000; Sponza and Cigal 2008). Previous work with fluorescence in situ hybridization (FISH) results indicated that Methanosarcina-like was the dominant methanogenic archaea present during PCP degradation experiments (Heimann et al. 2006). Under methanogenic culture, PCP was dechlorinated by acetoclastic methanogens of Methanosarcina, oxidizing acetate into methane and carbon dioxide (Stuart et al. 1999; Montenegro et al. 2003), suggesting that Methanosarcina was directly associated with PCP dechlorination.

Notably, besides the archaea, the bacteria also seem to play important roles in PCP dechlorination in the transferred soil/acetate suspensions. Anaeromusa and Syntrophobotulus are in the Clostridia class and associated with the Veillonellaceae and Peptococcaceae family, respectively, which contain many PCP-dechlorinating bacteria (Ruckdeschel et al. 1987; Tartakovsky et al. 2001; Chen et al. 2012). In a previous study, Veillonellaceae and Peptococcaceae bacteria have been disclosed the function for iron reduction coupling PCP dechlorination (Tong et al. 2014). Veillonellaceae are strictly anaerobic gram-negative cocci, which contain many iron reducing species (Li et al. 2011), whereas Peptococcaceae contains the well-known reductive dechlorinating bacterial genera Dehalobacter which can reduce PCP in soil suspensions with biochar and lactate (Tong et al. 2014). In our study, the enriched microcosm was preferable dechlorination in ortho-position following to meta- then para-positions, which was consistent with the pathway of dechlorination by some dechlorinating bacteria belonged to Peptococcaceae family (Christiansen and Ahring 1996; Utkin et al. 1995). The iron reducing microorganisms may be capable of using PCP as electron acceptors directly or they may first donate electrons to Fe(III), and then PCP was dechlorinated by Fe(II) generated from Fe(III) reduction (McCormick and Adriaens 2004). The aforementioned discussions suggested the important roles of Syntrophobotulus and Anaeromusa in iron reduction coupling PCP dechlorination. In our experiment, the soil contained 20.4 g kg−1 total Fe, and the content of Fe gradually declined with increased cycle transferred. Thus, a lower amount of Fe was supplied for microorganism respiration as cycles increased, which was concordant to the change of relative abundance of Veillonellaceae (genera Anaeromusa, Fig. 3). Also, a similar tendency existed in the variation of relative abundance of Anaeromusa and the PCP dechlorination rate (Figure S4), which indicated Anaeromusa directly involved in PCP dechlorination.

The relative abundance of Zoogloea, belonging to the Rhodocyclaceae family of β-Proteobacteria, achieved maximum value of 21.5% at the 4th cycle. Rhodocyclaceae strains have been described as having the high reduction abilities, not only for dechlorinating chlorinated compounds (Bae et al. 2007; Guan et al. 2013), but also in NO3 − reduction and organic contaminant degradation (Li et al. 2005; Shao et al. 2009). In the recent study, Rhodocyclaceae was identified as PCP degraders with lactate as carbon source, and the dechlorination pathway was consistent with our present study (Tong et al. 2015), indicated that Zoogloea may directly participate in dechlorination in ortho-position (2,3,4,5-TeCP was detected in our experiments). In addition, Zoogloea species isolated from contaminated groundwater were able to degrade 2,3,4,6-TeCP and PCP (Männistö et al. 1999). At circumneutral pH, Zoogloea has been reported as anaerobic Fe(II) oxidizer in iron redox cycling cultures (Coby et al. 2011; Li et al. 2016). Because Zoogloea was rarely found in the initial soil sample but became increasingly enriched in the treatments during PCP dechlorination in different cycles, it most likely acclimated to the transferred cycle cultures and involved in iron cycle and PCP degradation.

The relative abundances of Treponema, W22, and unclassified Cloacamonales continuously increased with the increasing cycle. Treponema, belonged to the Spirochaetaceae family, has been detected in many dechlorination cultures (Duhamel and Edwards 2007; Miura et al. 2015). However, it should be noted that none direct evidence has been ever provided to link the function of Treponema with PCP dechlorination. In dechlorination cultures, Treponema acts as a homoacetogen that is able to ferment carbon sources to hydrogen, carbon dioxide, and formate, which are essential for other dechlorinating bacteria (Graber et al. 2004; Miura et al. 2015). In the experiments, a significant positive liner correlation was observed between the relative abundance of Treponema and PCP dechlorination rate (R 2 = 0.59) (Figure S5). The dechlorination rate of PCP increased when with a higher relative abundance of Treponema. Thus, the fast PCP dechlorination and increased of relative abundance of Treponema during transferred cycles, suggest that Treponema plays important roles in the PCP dechlorination and may be a key syntrophy required for dechlorinating bacteria to dechlorinate PCP. Compared to the proportion of Treponema, the relative abundances of W22 and unclassified SHA-116, belonged to the Cloacamonales order, occupied a lower percentage of the culture. Cloacamonales has been previously described as a new bacterial phylum, WWE1 branching, and has been widely observed in anaerobic ecosystems (Riviere et al. 2009; Lucas et al. 2015), while not being reported to be able to use chlorophenols as the terminal electron acceptor. In the anaerobic sludge digester, Cloacamonales plays important roles in organic waste and wastewater treatment (Chouari et al. 2005; Narihiro and Sekiguchi 2007). The enrichment of Cloacamonales in the dechlorination cultures indicated that Cloacamonales may participate in syntrophic acetate-oxidization coupled with the dechlorination of PCP.

Based on the above mentioned PCP dechlorination, input consumption, and the microbial communities involved, an overall hypothesis can be proposed as Fig. 5. In the incubation cultures, Syntrophobotulus, Anaeromusa, and Zoogloea play dominant roles in iron cycle coupled to PCP dechlorination, whereas, Methanosarcina may be the dominant genera for direct PCP dechlorination coupled to organic matter oxidation to carbon dioxide and methane. With the input of organic carbon (acetate), Treponema, W22, and unclassified Cloacamonales became the dominant genera for direct or syntrophic function of PCP dechlorination. Acetate is the most important metabolite of the anaerobic food chain in rice field soils and thermodynamically feasible to oxidize to CO2 when it is coupled to iron reduction or syntrophic acetate-oxidizing consortia (Hori et al. 2010). In addition, the iron cycle process with high iron contents from southern China coupled with acetate oxidation can be strongly influenced the reductive dechlorination (Chen et al. 2012; Xu et al. 2015). Our results obtained from the microcosm experiments only revealed the dominant PCP-respiring microorganisms collected in the cycle culture. Further studies are needed to explore the function of acetate and the mechanism of direct acetotrophic PCP dechloriantion, which would be help for the bioremediation of iron rich paddy soil with organic chloride pollutants. However, a highly enriched culture or a pure culture is require, and efforts are made in our lab in this direction (e.g. isolation and additional testing).

Fig. 5
figure 5

The hypothesis of the microbial communities involved in the processes of PCP dechlorination in the cultures

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Conclusion

Our experiments show that a microbial system undergoing repeated cultivations in the input of organic substrate and PCP, develop a community structure capable of PCP dechlorination under anaerobic conditions. Anaeromusa, Syntrophobotulus, and Zoogloea were found to be the dominant genera in an iron cycle-associated PCP dechlorination. The Methanosarcina associated with humic substances and organic substrate, was enriched in the PCP dechlorination culture at different cycles. Treponema, W22, and unclassified Cloacamonales were dominant in the subsequent input of organic substrate, which expanded the diversity of microbial communities associated with PCP dechlorination. The more cycles of PCP dechlorination, the more stable the microbial communities became. The results of this study have important implications for shelling insight on understanding of microbial and geochemical interactions in the transformation of organic contaminants in iron rich soil.