Environ Sci Pollut Res (2016) 23:23184–23194 DOI 10.1007/s11356-016-7562-8
RESEARCH ARTICLE
Shifts in indigenous microbial communities during the anaerobic degradation of pentachlorophenol in upland and paddy soils from southern China Yating Chen 1,2,3 & Liang Tao 2 & Ke Wu 2,4 & Yongkui Wang 2
Received: 19 May 2016 / Accepted: 30 August 2016 / Published online: 6 September 2016 # Springer-Verlag Berlin Heidelberg 2016
Abstract Pentachlorophenol (PCP) is a common persistent pesticide in soil that has generated a significant environmental problem worldwide. Therefore, anaerobic degradation of PCP by the soil indigenous microbial community has gained increasing attention. However, little information is available concerning the functional microorganisms and the potential shifts in the microbial community associated with PCP degradation. In this study, we conducted a set of experiments to determine which components of the indigenous microbial community were capable of degrading PCP in soils of two land use types (upland and paddy soils) in southern China. Our results showed that the PCP degradation rate was significantly higher in paddy soils than that in upland soils. 16S ribosomal RNA (rRNA) high-throughput sequencing revealed significant differences in microbial taxonomic composition between the soil with PCP and blank (soil without PCP) with Acinetobacter, Clostridium, Coprococcus, Oxobacter, and
Sedimentibacter dominating the PCP-affected communities. Acinetobacter was also apparently enriched in the paddy soils with PCP (up to 52.2 %) indicated this genus is likely to play an important role in PCP degradation. Additionally, the Fe(III)-reducing bacteria Clostridium may also be involved in PCP degradation. Our data further revealed hitherto unknown metabolisms of potential PCP degradation by microorganisms including Coprococcus, Oxobacter, and Ruminiclostridium. Overall, these findings indicated that land use types may affect the PCP anaerobic degradation rate via the activities of indigenous bacterial populations and extend our knowledge of the bacterial populations responsible for PCP degradation. Keywords Subtropical soils . Land use type . Microbial degradation . High-throughput sequencing . Indigenous microorganisms . Acinetobacter
Responsible editor: Robert Duran Electronic supplementary material The online version of this article (doi:10.1007/s11356-016-7562-8) contains supplementary material, which is available to authorized users. * Liang Tao
[email protected]
1
Guangzhou Institute of Geochemistry, Chinese Academy of Sciences, Guangzhou 510640, People’s Republic of China
2
Guangdong Key Laboratory of Agricultural Environment Pollution Integrated Control, Guangdong Institute of Eco-Environmental and Soil Sciences, Guangzhou 510650, People’s Republic of China
3
University of Chinese Academy of Sciences, Beijing 100049, People’s Republic of China
4
College of Chemistry and Chemical Engineering, Hunan Normal University, Changsha 410081, People’s Republic of China
Introduction Pentachlorophenol (PCP) is an organic compound widely used in both industry and agriculture as a pesticide, herbicide, and wood preservative (Czaplicka 2004). It is known to bioaccumulate, to be very toxic, and to generate harmful consequences in soil and aquifer environments because of its low biodegradability and chemical stability (Puglisi et al. 2009; Li et al. 2015). Therefore, searching for a cost-effective and environmentally friendly approach to eliminate this contaminant is now a worldwide priority. When PCP enters the soil, it undergoes a slow natural dissipation (Chen et al. 2012), and the biodegradation of PCP has been proposed to be the most important PCP removal process in soils (Kim et al. 2004; Yoshida et al. 2007; Field and SierraAlvarez 2008; Juwarkar et al. 2010; Bosso and Cristinzio
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2014). Essentially, in the anaerobic environment, PCP degradation is a microbial dechlorinated respiration process, during which the chlorinated compounds serve as terminal electron acceptors (Saia et al. 2007; Payne et al. 2011). In our previous study, Chen et al. (2012) revealed that the dechlorinated products of degradation of PCP are 2,3,5,6-tetrachlorophenol (TeCP) and 2,3,5-trichlorophenol (TCP), and Tong et al. (2014) also found the similar intermediates in dechlorination of PCP. According to these reports, PCP is expected to be dechlorinated under anaerobic conditions by microbes at the ortho-position (Bouchard et al. 1996; Field and Sierra-Alvarez 2008). Recently, cultivation-independent methods have been widely used to profile the microorganisms that specifically degrade PCP and chlorophenols in soils (Montenegro et al. 2003; Mahmood et al. 2005). Yoshida et al. (2007) enriched a microbial community from paddy soil that degraded PCP, and the structure of the microbial community was characterized by fluorescence in situ hybridization and PCR-DGGE (denaturing gradient gel electrophoresis). Chen et al. (2012) used the terminal restriction fragment length polymorphism (T-RFLP) method to study the anaerobic degradation of PCP by indigenous microorganisms in paddy soils collected from typical sites in southern China. Furthermore, DNA-based stable isotope probing (SIP) was applied to explore the key microorganisms responsible for PCP mineralization in paddy soils (Tong et al. 2015; Li et al. 2015). Despite these important advances, we still have relatively limited knowledge on the real composition and diversity of the microbial community during PCP degradation in soils, which are considered to be the most diverse microbial habitats on Earth (Gans et al. 2005; Janssen 2006). Fortunately, Illumina high-throughput sequencing has enabled the in-depth exploration of microbial biodiversity in the environment (Nacke et al. 2011; Chen et al. 2014a). This technology is capable of significantly expanding the detection range of low-abundance taxas in the community, which are generally not available by conventional molecular methods. Moreover, most previous studies attempted to investigate soil microbial communities at the conclusion of PCP degradation or at a specific time point during PCP degradation, resulting in the poor understanding of the succession of the microbial community. This understanding would aid in the evaluation of the potential for nature attenuation. Soil in southern China has a great potential for rice cropping because of the subtropical monsoon climate in this region (Liu 1993; Lu and Shi 2000; Lin et al. 2004). However, the area of land used as upland for vegetable and fruit cultivation has increased yearly (Lin et al. 2004). Hence, the influence of land use types on microbial degradation of PCP cannot be underestimated. In the present study, we applied highthroughput sequencing to profile the changes in diversity and abundance of microbial communities during the anaerobic degradation of PCP in soils of two different land use types (upland and paddy soils) and to address the potential functional microorganisms during PCP
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biodegradation. Experiments were conducted to perform the following tasks: (1) evaluate and compare the PCP degradation rates in upland and paddy soils; (2) investigate the shifts in microbial community structures derived from upland and paddy soils during the anaerobic degradation of PCP; and (3) explore the potential functional microbial groups during PCP-anaerobic degradation.
Materials and methods Soil sampling and chemicals The six soil samples used in the present study were collected in Guangdong Province, China; the classification and the sampling locations of the soils are shown in Table S1 in Supporting Information (SI). At each sampling site, soils were collected from the surface layer (0–20 cm). Four subsamples with 25 × 25 cm surfaces were collected and then mixed to obtain a bulk sample. The sampled soils were stored in ice boxes and immediately transported for storage at 4 °C prior to the batch experiments. We divided the samples into two groups (upland and paddy soils) according to the types of land use. All chemicals used were of analytical grade or above. PCP (≥98 % purity) and piperazine-N, N′-bis-(2-ethanesulphonic acid) (PIPES, ≥99 % purity) were obtained from SigmaAldrich (St. Louis, MO, USA). The PCP stock solution (94 μM) was prepared by dissolving PCP in a 1.0 M NaOH solution. Geochemical characteristics The samples were air-dried naturally and passed through a 2mm sieve for subsequent analysis. The pH was measured at a 1:2.5 (w/v) sample-to-distilled water ratio. The organic matter content was determined by the potassium dichromate heating oxidation method. The total concentrations of Fe, K, Na, and Al were determined by atomic absorption spectrophotometry on an inductively coupled plasma atomic emission spectrometer after digestion with an HClO4/HF/HNO3 mixture (Pansu and Gautheyrou 2006) and are reported as the equivalent oxide contents (Fe2O3, K2O, Na2O, and Al2O3, respectively). The CaO and MgO contents of the soils were measured using the Na2CO3 melting-mass method (Lu 1999). Batch experiments The typical reduction experiments were performed in 20-mL serum bottles with silicone-lined septa and aluminum sealing caps. Each reactor contained 0.5 g soil (dry weight), along with 10-mL PIPES buffer (30 mM) solution to maintain a constant pH of 7.0 ± 0.2, and PCP was added at a final concentration of 5.0 mg L−1. This concentration was normalized
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to 100 ng g−1 soil when mixed with 0.5 g soil in the study, which is in the range of residue concentrations of PCP in soils of Guangdong Province (Hong et al. 2005; Zheng et al. 2012). After purging with O2-free N2 gas (99.99 %) for 30 min, the mixtures in the reactors were sealed and placed in an incubator set at 30 ± 1 °C in the dark. Triplicate samples were destructively sampled at each specific time point of incubation. For comparison, controls (blank) that PCP was not present in the reactors were also performed. All materials, including the serum bottles, butyl rubber stoppers, and solutions, were sterilized in an autoclave at 121 °C for 20 min prior to use. Analysis At each time point, the PCP in the soil suspension was extracted with a water/ethanol mixture at a volumetric ratio of 1:1 for 1 h on a horizontal shaker (180 rpm) (Chen et al. 2012). The water-ethanol-soil mixture was filtered through a 0.45-μm filter. The filtrate was collected for highperformance liquid chromatography (HPLC) analysis of the PCP concentration. Details of the HPLC analysis are provided elsewhere (Lan et al. 2008; Chen et al. 2012; Wang et al. 2012; Tong et al. 2014). Soil samples prepared for gas chromatography-mass spectrometry (GC-MS) analysis were pretreated as follows. Firstly, the samples were freeze-dried and then extracted with 30 mL of ethyl acetate for three times. Afterward, the extracts were dehydrated with anhydrous sodium sulfate, and were concentrated to 0.5 mL. A Cleanert C18-SPE column was used to purify the extracts, which were eluted with 4 mL of 1:1 (v/v) mixture of methyl alcohol and dichloromethane. Finally, the filtrate was concentrated to 0.5 mL using a rotary evaporator (41K30RA-C, Gongyi Yingyu Instruments Co., Ltd., Gongyi, Henan, China), followed by derivatization with the mixture of N,O-bis(trimethylsilyl)trifluoroacetamide and 1 % trimethylsilyl chloride. The PCP degradation intermediates were determined on a GC-MS spectrometry (GC-MS-QP2010 Plus, Shimadzu) equipped with DB-5MS column (30 m × 0.25 mm × 0.25 μm, Agilent J&W Scientific, Folsom, CA). The injector and ion source temperatures were 280 and 230 °C, respectively. The oven temperature was held at 60 °C, increased to 250 °C at a rate of 15 °C min−1, further increased to 280 °C at a rate of 3 °C min−1, and then increased to 300 °C at a rate of 5 °C min−1, and maintained for 0.5 min. DNA extraction, PCR amplification, and high-throughput sequencing The community DNA of microbes from soil enrichment culture samples was extracted at each designated time point using a PowerSoil DNA isolation kit (Mo Bio, Carlsbad, CA, USA) according to the manufacturer’s protocol. The V4 hypervariable
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region of the bacterial and archaeal 16S rRNA genes was amplified by PCR using the F515 and R806 primer set (Fierer et al. 2008) and a sample-specific 12-bp barcode on R806. Each DNA sample was amplified in triplicate in 30-μL reaction mixtures under the following conditions: initial denaturation at 94 °C for 5 min, 30 cycles of denaturation at 94 °C for 30 s, annealing at 50 °C for 1 min, and extension at 72 °C for 1 min, followed by a final extension period at 72 °C for 10 min. A single composite sample was prepared by combining equimolar amounts of PCR products from individual samples purified with a QIAquick Gel Extraction Kit (QIAGEN, Chatsworth, CA, USA) and sequenced using the Illumina MiSeq (paired-end 250-bp mode) platform (Illumina, San Diego, CA, USA).
Processing of 16S sequencing data The 16S ribosomal RNA (rRNA) MiSeq sequencing data were processed using Mothur and QIIME (Schloss et al. 2009; Caporaso et al. 2010). The raw reads were trimmed with a quality less than 20 and assembled into contigs. Chimeric sequences were also removed by the chimera.uchime command with the default parameter. 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. Because the minimum number of quality sequences per community was 15,800 across all 54 samples, calculations of alpha diversity (including Chao1, Faith’s PD, Shannon, and Simpson) was based on a subset of 15,800 randomly selected sequences from each sample.
Statistical analysis All statistical analyses were implemented using SPSS 18.0 and various R packages (http://www.r-project.org). The independent samples t test was performed to test the differences in soil properties, PCP reduction rates, and relative abundances of dominant lineages between the upland and paddy soils. One-way ANOVA was performed to test the differences in microbial characteristics during PCP degradation process under the different reaction times. Differences between means were tested by the least significant difference (LSD) method at the 5 % level. Pearson’s pairwise correlation analysis was used to assess the relationship among the PCP degradation rates, soil properties, and relative abundances of the dominant lineages in the upland and paddy soils.
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Results
SI). The obtained results proved the degradation processes of PCP underwent reductive dechlorination reaction under anaerobic conditions.
General characteristics of the soil samples The soil physiochemical characteristics are summarized in Table 1. In general, the soils were slightly acidic, with the exception of A-1, which was near neutral. The upland soils generally had lower pH values than the paddy soils (Table 1). Additionally, the soils had highly variable organic matter (OM) contents, with a range of 16 to 50 g kg−1 (average 30 g kg−1). The OM, total N (TN), and cation exchange capacity (CEC) were, on average, higher in the paddy soils than in the upland soils. The essential elements in soils (Al, Fe, K, Mg, Na, and Ca) were also determined (Table 1).
Anaerobic degradation of PCP in the upland and paddy soils The PCP degradation kinetics obtained in the upland and paddy soil suspensions are shown in Fig. 1. The dissipation of PCP in the two soils was modeled with a first-order kinetic model, and the values of the degradation rate constants (k), the relative coefficient (R2), and half-lives (t1/2) were calculated (Table 2). In the present study, the degradation of PCP in all upland soils was similarly low (3.14 to 4.69 %) with the reaction time of 40 days, with k values ranging from 7.88 × 10−4 to 1.28 × 10−3 day−1 and half-lives ranging between 542 and 880 days (Fig. 1a and Table 2). In contrast, the PCP degradation ratios in the paddy soils were overall higher than those in the upland soils (Fig. 1b). Nearly 100 % of the PCP was degraded after approximately 13–18 days in paddy soils with constants ranging from 4.79 × 10−2 to 1.19 × 10−1 day−1 and half-lives ranging between 5.81 and 14 days (Table 2). Using gas chromatography-mass spectrometry analysis, the complete time course of the anaerobic degradation shows the continuously increased concentration of tetrachlorophenol (TeCP) and trichlorophenol (TCP), with one and two chlorines dechlorinated in the PCP molecular structure, respectively, accompanied by PCP concentration decreasing (Fig. S1 in Table 1 Land use
Upland Upland Upland Paddy Paddy Paddy
Microbial community diversity and richness A total of 2,745,952 sequences were obtained for the 54 analyzed samples from the 16S rRNA MiSeq sequencing analysis of the microbial communities after filtering out poor-quality sequences, with a range of 15,817 to 193,652 sequences per community (Table S2 in SI). The number of unique and classifiable representative OTUs was 79,555 in total in the complete data set, with an average of 1473 OTUs per sample (Tables S3 and S4 in SI). The alpha diversity of the microbial community was evaluated based on 15,800 sequences randomly selected from each sample (Tables S3 and S4 in SI). The microbial diversity of soils with PCP were significantly higher (t test, all P < 0.05) than that in blank in both upland and paddy soils. However, there were no significant differences between the overall microbial diversity (Chao1, Faith’s PD, Shannon, and Simpson) in upland and paddy soils with PCP (t test, all P > 0.05). In comparison, the mean Chao1 richness of the microbial community in the paddy soils (4219 ± 354) was higher than that in the upland soils (3825 ± 317). Microbial community composition and differences Bacteria were predominant in the communities of all the soils samples with a relative abundance of 96.1 %, whereas only a small proportion of the sequences were assigned to Archaea. Of the classifiable 16S rRNA sequences, 24 bacterial phyla were identified across the sample set (Fig. 2). In communities of the upland soils (blank and soil with PCP), the most frequently detected were Firmicutes (13.3–72.1 % across all samples), Proteobacteria (9.2–43.9 %), Chloroflexi (0.8– 31.6 %), Planctomycetes (0.5–11.6 %), and Acidobacteria (0.6–10.3 %). For the Archaea, the majority of sequences were affiliated with Euryarchaeota (2.0 %) (Fig. 2a). In
Physicochemical characteristics of soil samples Sample
pH
OM (g kg−1)
TN (g kg−1)
CEC (cmol (+) kg−1)
Al2O3 (g kg−1)
Fe2O3 (g kg−1)
K2O (g kg−1)
MgO (g kg−1)
Na2O (g kg−1)
CaO (g kg−1)
G-1 B-1 L-1 A-1 A-2 A-3
4.44 4.75 4.56 6.23 5.20 5.40
16 39 30 50 23 21
0.9 1.4 2.1 3.3 0.4 1.1
7.0 9.9 17.4 21.6 4.9 7.9
249 275 179 185 62 150
75 175 63 81 16 41
27 3 26 24 10 25
4.8 0.9 10.5 10.2 1.7 3.7
2.3 2.3 8.1 4.7 3.0 4.5
0.8 1.1 2.2 8.1 2.0 2.3
OM organic matter, TN total nitrogen, CEC cation exchange capacity
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Fig. 1 Kinetics of PCP degradation in upland (a) and paddy (b) soils
communities of the paddy soils (blank and soil with PCP), the dominant taxas across all samples were Firmicutes (24.2– 87.7 %), Proteobacteria (2.3–66.7 %), Chloroflexi (0.7– 22.9 %), Planctomycetes (0.5–14.4 %), Acidobacteria (0.3– 17.2 %), and Crenarchaeota (0.3–5.8 %) (Fig. 2b). Taxonomic classification revealed obvious differences in community composition between soil with PCP and the corresponding blank in both upland and paddy soils (Fig. 2). Specifically, in upland soils, Proteobacteria was significantly more abundant in soil with PCP than in blank (t test, P < 0.05), whereas the relative abundance of Firmicutes and Acidobacteria showed opposite results. In paddy soil, the microbial composition revealed broadly similar distribution patterns of dominant bacterial taxas, and the relative abundance of Proteobacteria was significantly higher in soil with PCP (47.6 %) than that in blank (2.6 %) (t test, P < 0.05). The distribution of dominant taxa also varied between the upland and paddy soils with PCP. The dominant taxas in the upland soils with PCP were Firmicutes (46.2 ± 7.3 %), Proteobacteria (31.6 ± 6.6 %), and Chloroflex (7.7 ± 3.6 %), while in the paddy soils with PCP, the predominant phylogenetic group was Proteobacteria (47.6 ± 9.7 %), followed by Firmicutes (39.1 ± 4.8 %) and Chloroflex (5.6 ± 2.1 %) (Figs. 2 and S2 in SI). Furthermore, the relative abundance of dominant taxas also varied considerably across the PCP anaerobic degradation in both upland and paddy soils (Fig. S2 in SI). In particular, the relative abundance of Chloroflex, Alphaproteobacteria, Actinobacteria, Planctomycetes, and Acidobacteria significantly decreased in paddy soils with PCP, suggesting that these taxas were possibly not adapted to the pollution stress of PCP. In contrast, the relative abundance of Gammaproteobacteria increased as the PCP degradation progressed (Fig. S2b in SI), Table 2 First-order rate constants (k) and half-lives (t1/2) of PCP transformation in upland and paddy soils
indicating the important roles of Gammaproteobacteria in PCP degradation. Similarly, the dominant archaea Euryarchaeota presented increasing trends in PCP degradation in both upland and paddy soils, with the relative abundance increasing from 1.3 to 8.9 and 1.2 to 5.0 %, respectively (Fig. S2 in SI). At the genus level, in the original upland soils, the dominant indigenous genera were Acidiphilium of class Alphaproteobacteria, Pseudomonas and Acinetobacter of class Gammaproteobacteria, Clostridium and Bacillus of phylum Firmicutes, and Anaerolinea of phylum Chloroflexi. While in paddy soils, members from Clostridium, Bacillus, Acinetobacter, Anaerolinea, and Paenibacillus of phylum Firmicutes dominated the indigenous microbial communities (Fig. S3 in SI). Furthermore, the microbial community composition differed considerably across the blank and soil with PCP (Fig. S3 in SI). In the cultures of blank, Symbiobacterium of phylum Firmicutes was the most dominant genus in both soils, although other representative genera of phylum Firmicutes, such as Acetivibrio, Thermofilum, Sporomusa, and Clostridium, were also frequently detected. Correspondingly, Acinetobacter was the most dominant genus in cultures of soil with PCP, followed by Clostridium, Acidiphilium, and Methanosarcina of phylum Euryarchaeota, and Caloramator and Coprococcus of phylum Firmicutes, while the well-known reductive dechlorinationors (Desulfitobacterium and Dehalobacter of phylum Firmicutes, and Dehalococcoides of phylum Chloroflexi) were rarely detected. As the incubation progressed, the dominant microbial genera also displayed distinct succession under different treatments (Figs. 3 and S4 in SI). The relative abundance of Symbiobacterium increased within the 30-day incubation in blank. By contrast, the relative abundance of Acinetobacter, Clostridium, Methanosarcina, Coprococcus, Oxobacter, and
Upland Sample G-1 B-1 L-1
Paddy k (day−1)
R2
t1/2 (day)
Sample
k (day−1)
R2
t1/2 (day)
1.28 × 10−3 1.02 × 10−3 7.88 × 10−4
0.958 0.928 0.665
542 680 880
A-1 A-2 A-3
1.19 × 10−1 9.50 × 10−2 4.79 × 10−2
0.813 0.645 0.743
5.81 7.29 14
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Fig. 2 Relative abundance (%) of dominant microbial taxa across upland (a) and paddy (b) soils at different reaction time (controls (blank) vs. soil with PCP). Other includes Armatimonadetes, SC4, Verrucomicrobia,
Gemmatimonadetes, Cyanobacteria, WPS-2, AD3, Nitrospirae, and Chlorobi. The phyla with a relative abundance >1 % in at least one sample were defined as dominant phyla
Sedimentibacter dramatically increased in soils with PCP (Fig. S4 in SI). Notably, the relative abundance of these genera significantly increased as the enrichment progressed in paddy soils with PCP (Fig. 3).
greatly stimulated by PCP during the degradation in paddy soils. Taken together, the present study reveals that land use types affect the reductive degradation of PCP through the activities of indigenous microbes, and Acinetobacter plays an important role in PCP degradation in soils.
Discussion
PCP degradation correlated to soil properties
The present study assessed the impact of land use types (upland and paddy soils) on the degradation of PCP in subtropical soils and investigated the shifts in the microbial community structure and potential functional microorganisms during the anaerobic degradation of PCP using deep 16S rRNA sequencing. During soil incubations with PCP, the PCP degradation was more efficient in paddy soils than that in upland soils. 16S rRNA gene-based sequencing revealed the potential shifts of bacterial populations in all blank and soil with PCP treatments. Acinetobacter was identified as the most dominant genera in communities of soil with PCP. This genus was also
The detailed relationships between the first-order rate constants (k) and soil properties were examined with Pearson’s pairwise correlation test (Table 3). Here, the coefficients showed a positive correlation between the first-order rate constant of PCP degradation in upland soils (k1) and the initial Al2O3, Fe2O3, and K2O, and a negative correlation with OM, TN, CEC, MgO, Na2O, and CaO (Table 3). In paddy soils, all soil properties showed positive correlation between the firstorder rate constant of PCP degradation (k2), with the exception of K2O and Na2O (Table 3). However, the results of correlation analysis showed no significant correlations among PCP
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Fig. 3 Relative abundance (%) of dominant microbial genera of the enrichment cultures with PCP in upland (a) and paddy (b) soils. Within each group, bars with different lowercase letters were significantly different from each other (P < 0.05, LSD)
degradation and soil properties. These results were in accordance with other surveys investigating the PCP degradation under anaerobic conditions (Lin et al. 2012; Chen et al. 2014b). It has been widely reported that the solubility of the PCP anion increases significantly with the increasing pH and that the absorption of PCP by organic matter is also dependent on the soil pH (Arcand et al. 1995; McAllister et al. 1996; He et al. 2006). In this study, the pH value was kept constant in Table 3 Correlation analysis of PCP degradation rates with physicochemical properties k1
k2
Pearson
P
Pearson
OM TN CEC Al2O3 Fe2O3
−0.630 −0.992 −0.960 0.681 0.065
0.566 0.082 0.180 0.523 0.959
0.798 0.588 0.638 0.094 0.453
0.412 0.600 0.559 0.940 0.700
K2O MgO Na2O CaO
0.070 −0.563 −0.849 −0.939
0.956 0.619 0.354 0.224
−0.243 0.593 −0.078 0.730
0.844 0.596 0.951 0.479
the reactors to exclude the effects of soil characteristics on PCP degradation. In support of this, the correlation analysis showed no significant correlations among PCP dissipation and soil properties in both upland and paddy soils (Table 3). In addition, PCP is mostly present as the more soluble phenolate ion at neutral pH (Scelza et al. 2008), which is considered to be the bioavailable fraction for microorganisms (MataSandoval et al. 2001). The optimum pH range for the activity of most microorganisms ranges from approximately 6.0 to 7.5 (McAllister et al. 1996), highlighting the possibility that the biodegradation caused by indigenous microorganisms represents the important soil PCP removal process.
P
k1 k under upland soils, k2 k under paddy soils; other abbreviations are same as Table 1
Microbial community shifts corresponded with PCP degradation The phylogenetic diversity of soil microbial communities has been frequently reported to be closely associated with the soil pH on both small and large scales, and the relative abundances of microbial groups at different taxonomic levels responded strongly to the soil pH (Fierer and Jackson 2006; Hartman et al. 2008; Lauber et al. 2009; Rousk et al. 2010). In this study, to eliminate the effect of pH on the structure and diversity of the microbial community, we added a buffer solution to the reactors to keep the pH value at 7.0. This step allowed us to
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better assess the ability of indigenous microorganisms to degrade PCP. 16S rRNA-based high-throughput sequencing results from the blank and soil with PCP incubations reveled that PCP degradation alters the structures of indigenous bacterial populations in both upland and paddy soils (Figs. 2 and 3). As the incubation progressed, Symbiobacterium belonging to Firmicutes was obviously enriched in blank and the relative abundance of this genus increased sharply to an average value of 20.5 %. In fact, Symbiobacterium has been found in our previous study of PCP degradation, and showed predominance in the blank treatment as well (Chen et al. 2014b). By contrast, the relative abundance of Acinetobacter, Clostridium, Methanosarcina, Coprococcus, Oxobacter, and Sedimentibacter dramatically increased in soils with PCP (Figs. 3 and S4 in SI). In particular, in upland soils, Acinetobacter (the fifth most abundant genus in the original soils) became the most dominant genus within the 10-, 20-, and 30-day incubation with a highest relative abundance of 20.8 % at day 20. The relative abundances of Clostridium, Methanosarcina, and Oxobacter evidently increased to an average value of 12.3, 2.6, and 2.3 %, respectively, during PCP degradation (Figs. 3a and S4 in SI). In paddy soils, the relative abundance of Acinetobacter, Coprococcus, Clostridium, and Oxobacter sharply increased to an average value of 54.7, 7.3, 6.3, and 1.4 %, respectively, within 20-day incubation. While the relative abundance of Acinetobacter and Coprococcus decreased dramatically to 22.3 and 4.3 % after 20-day incubation. Surprisingly, the relative abundance of the dominant genera was not significantly (P > 0.05) different in the PCP degradation (Fig. 3a) in the upland soils. However, the relative abundance of Acinetobacter, Clostridium, Caloramator, Coprococcus, Methanosarcina, Oxobacter, and Sedimentibacter significantly increased as the degradation progressed in the paddy soils (Fig. 3b). Among the active bacterial population detected in soil with PCP treatment, Acinetobacter was of interest because of the apparent enrichment in both soils (Fig. 3). In our study, PCP was completely degraded after approximately 20 days in paddy soils (Fig. 1), and the relative abundance of Acinetobacter appeared to be corresponded with the PCP degradation (Fig. S4 in SI). In addition, the distribution of this genus also showed significant differences between the upland and paddy soils with PCP (t test, P < 0.05) (Fig. S5 in SI). These results suggested that Acinetobacter might play a critical role in PCP degradation in paddy soils. Members of Acinetobacter have been defined as PCP degraders (Stanlake and Finn 1982; Sharma and Thakur 2008; Sharma et al. 2009), they have the ability to utilize PCP through an oxidative route with ortho ring-cleavage by producing intermediate products like 2,3,5,6-tetrachlorohydroquinone (TCHQ) and 2-chloro-1,4-benzenediol (DCBE) (Sharma and Thakur 2008; Sharma et al. 2009). Interestingly, in this study, we found that the primary intermediates of reductive degradation of PCP were TeCP and TCP in the anaerobic reactors,
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suggesting the possibility that Acinetobacter was able to degrade PCP through the reductive route when presented in the soilmicrobe complex. It is also noteworthy that Clostridium was detected as the dominant genus accounting for up to 15.6 % of bacterial communities in soils with PCP (Fig. S4 in SI). The Fe(III) reducing bacteria Clostridium was also observed in the anaerobic enrichment with PCP degradation (Xu et al., 2015). Members of this genus were able to degrade PCP via pH modification by Fe(III) compounds or increase the degradation rate of PCP by generating sorbed Fe(II) (Xu et al., 2015). In this study, considering the high concentration of Fe2O3 in both soils (Table 1), it is assumed that Fe(III)-reducing bacteria Clostridium might be involved in PCP degradation. Our results suggested the microbes that involved in PCP degradation might be far more complicated than previously. For example, Coprococcus, Oxobecter, and Ruminiclostridium revealed significant negative correlations with the concentration of PCP in the reactors (Fig. S6 in SI), but there was no direct evidence thus far about their involvement in PCP degradation. Coprococcus and Ruminiclostridium have always been reported to produce acetate via fermentative metabolisms (Tsai and Jones 1975; Patel et al. 1981; Nissila et al. 2011; Zhang et al. 2014). The presence of this metabolic product might enhance the degradation rate of PCP (Chang et al. 1995). In addition, Oxobecter was detected in the anaerobic degradation of 1,1,1-trichloro-2,2bis (p-chlorophenyl) ethane (DDT) in paddy soils (Chen et al. 2013), indicating a possible organic pollutant degrading capacity of this genus. Land use effects on PCP degradation rates In this study, one striking feature of the PCP degradation rate phenomena observed for the six soils is that the fist-order rate constants (k) of PCP degradation were significantly higher in paddy soils than those in upland soils (t test, P < 0.05) (Table 2). Similar results were observed in other surveys, where significant differences in the degradation rates of PCP were observed under different land use types and the PCP degradation was more efficient in paddy soils than in upland soils (Chen et al. 2014b). Land use activities are human factors that not only add landscape-scale heterogeneity of the Earth’s surface, but also alter the microscopic features including microbial communities in soil (Girvan et al. 2003; Acosta-Martinez et al. 2008; Foster et al. 2009). Paddy soils are highly modified by anthropogenic activities that anoxic conditions prevail during most of the time of rice growth (Senesil et al. 1999; Kögel-Knabner et al. 2010). By contrast, uplands have distinctly different biogeochemical characteristics and aerobic conditions are sustained over a long period of time when upland crops are grown (Zhang and Gong 2003; Dubinsky et al. 2010). This might bring the different PCP degradation capacity and microbial communities. While reductive
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dechlorination has been considered to be the most effective step to degrade PCP in soil (Krooneman et al. 1999) and this process could only occur under anaerobic conditions. Therefore, in this study, we conducted anaerobic incubation to study the PCP degradation in both soils. Although the degradation of PCP in upland soils was slower than in paddy soils, the dominant microbes (like Acinetobacter, Clostridium, Coprococcus, Oxobacter, and Sedimentibacter) in the indigenous communities were also apparently enriched in the anaerobic incubation with upland soils, which may potentially play a role in reductive dechlorination of PCP in upland soils once the soils are under oxic conditions. Taken together, it is assumed that the divergences of PCP degradation rates observed in soils of different land use types might result from the activities of indigenous microbial communities.
Conclusions In summary, our results revealed that land use types may affect the PCP degradation rate via the activities of indigenous bacterial populations. Added PCP in soils increased the relative abundance of Acinetobacter, Clostridium, Coprococcus, Oxobacter, and Sedimentibacter. Furthermore, Acinetobacter and Coprococcus also had an apparently higher relative abundance in the paddy soils than upland soils with PCP. Therefore, the genus Acinetobacter may play an important role in PCP degradation in paddy soils. This genus may degrade PCP through the anoxic reductive dechlorinating pathway, which has not been reported before. Additionally, the Fe(III)-reducing bacteria Clostridium was likely to involved in PCP degradation. Furthermore, some of the Proteobacteria- and Firmicutes- related bacteria not known as PCP degraders to date may also be involved in PCP dechlorination. It should be pointed out that the results presented in this study were obtained under laboratory conditions that may deviate from the reality of field conditions. Therefore, future studies may focus on searching for specific microflora collaboration specifically suitable for remediation of soils contaminated with polychlorinated compounds under field conditions. Acknowledgments We thank Dr. Zhengshuang Hua and Dr. Haoyue Shu of Sun Yat-sen University for their help with the statistical analysis. This work was financially supported by the National Nature Science Foundation of P. R. China (no. 41271248), Guangdong Natural Science Funds for Distinguished Young Scholars (no. 2016A030306019), the National Key Basic Research Program of P. R. China (no. 2014CB441002) and the China Scholarship Council (file no. 201608440014).
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