Article pubs.acs.org/JAFC
Vertical Profiles of Pentachlorophenol and the Microbial Community in a Paddy Soil: Influence of Electron Donors and Acceptors Jiajiang Lin,†,‡ Yan He,*,† Jianming Xu,*,† Zuliang Chen,‡ and Philip C. Brookes† †
Institute of Soil and Water Resources and Environmental Science, Zhejiang Provincial Key Laboratory of Subtropical Soil and Plant Nutrition, Zhejiang University, Hangzhou 310058, China ‡ School of Environmental Science and Engineering, Fujian Normal University, Fuzhou 350007, China S Supporting Information *
ABSTRACT: Vertical variations of pentachlorophenol (PCP) dissipation and microbial community were investigated in a paddy soil with the addition of electron acceptors (NO3−, SO42−) and donors (crop residues). Crop residues enhanced PCP dissipation by supplying dissolved organic carbon (DOC) as an electron donor, whereas NO3− and SO42− inhibited it. The dissipation of PCP in electron donor treatments resulted in the accumulation of 3,4,5-trichlorophenol (3,4,5-TCP) except for wheat residues. The abundance and diversity of phospholipid fatty acids (PLFAs) decreased with increasing soil depth. The succession of predominant PLFAs shifted from aerobic bacteria to anaerobic bacteria when electron acceptors were changed to electron donors. The saturated/monounsaturated fatty acids (S/M) ratio increased with soil depth, which probably implied that nutrient turnover rate declined after the accumulation of 3,4,5-TCP. The results showed that the addition of electron donors and acceptors modified the microbial communities, which then further influenced the degradation pathway of PCP. KEYWORDS: pentachlorophenol (PCP), soil profiles, electron acceptors and donors, intermediate, phospholipid fatty acids (PLFAs)
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INTRODUCTION Pentachlorophenol (PCP) has been extensively used in China in fighting against snail fever and also as a herbicide since the 1970s.1,2 The International Agency for Research on Cancer (IARC) classifies PCP in group 2B, having inadequate evidence of human carcinogenicity and sufficient evidence for animal carcinogenicity.3 Pentachlorophenol is relatively persistent in soils and undergoes a slow natural dissipation.2 In soil, the dissipation of PCP is attributed to chemical, photochemical, and microbiological processes.4,5 The biodegradation of chlorinated phenols in the natural environment has been considered in many studies and may occur under both aerobic and anaerobic conditions.5,6 Under aerobic conditions, the biodegradation of PCP has typically been ascribed to monooxygenase and dioxygenase.7 Chlorohydroquinones are the initial intermediates of aerobic polychlorinated phenol biodegradation.6 Aerobic biodegradation of lower chlorinated phenols is rapid, but it is often slow for highly polychlorinated phenols. Under anaerobic conditions, PCP could be dissipated through dechlorination. Many anaerobic bacteria dechlorinate PCP at the ortho position.6,8 Only bacteria from the species Desulfitobacterium hafniense can reductively dechlorinate PCP completely, having enzyme systems for ortho, meta, and para dechlorination.8,9 Reductive dechlorination of organic contaminants can be affected by many redox processes such as the reduction of NO3−, SO42−, and Fe(III).4,5 The application of NO3−, SO42−, and Fe(III), which serve as electron acceptors, can compete for electrons with PCP dechlorination,4,10 whereas the organic matter serving as electron donors can improve it.9 Our previous studies showed that the addition of SO42− significantly inhibited DOC release, whereas NO3− increased it.11 Coates et al.12 confirmed that the quinone moieties are the redox-active components of the humic substance © 2014 American Chemical Society
for these microbial reductive reactions. In addition to these two processes, the adjacent aerobic and anaerobic zones at the soil−water interface were very common in wetland soils.7 Many common soil constituents undergo sequential oxidation and reduction reactions at adjacent aerobic and anaerobic zones.7 For example, Hall and Silver13 demonstrated that the anaerobic/ aerobic transitions characteristic of humid environments could stimulate organic matter decomposition. Our previous studies also showed increased DOC production under alternate wetting and drying cycles.11 However, there is scant information about the dechlorination pathway of PCP associated with the electron donors and acceptors at the adjacent aerobic and anaerobic zones in paddy soils. Crop residues have been widely added to soils as amendments. When submerged, they can be decomposed by microorganisms into various substances, including electron donors and carbon sources.14−16 Rice husk, pig farm wastewater treatment sludge, and coconut husk chips are also supplied to the soil as electron donors for sulfate-reducing bacteria, with a SO42− removal efficiency of 59%.17 Bi et al.15 found that the electron donor capacities of reducing dissolved organic matter from green manure were much higher than those from rice straw. The effect of electron donors and acceptors on organic pollutants in the environments where aerobic and anaerobic coexistence occurs remains unclear. Phospholipid fatty acids (PLFAs) are specific components of cell membranes found only in viable cells.18 PLFAs have been used to characterize microbial community structure changes in Received: Revised: Accepted: Published: 9974
June 12, 2014 September 24, 2014 September 25, 2014 September 25, 2014 dx.doi.org/10.1021/jf502746n | J. Agric. Food Chem. 2014, 62, 9974−9981
Journal of Agricultural and Food Chemistry
Article
was thereafter vented for 24 h for the methanol to vaporize and then mixed thoroughly with a large portion of uncontaminated soil (1:19, w/w). Then, the NaNO3, Na2SO4, and crop residues were added to the PCP-polluted soils at 1.7, 2.8, and 15 g kg−1 soil, respectively, and mixed thoroughly to establish eight treatments: (A) PCP unspiked control (CK1); (B) PCP without other addition (CK2); (C) PCP + NaNO3; (D) PCP + Na2SO4; (E) PCP + wheat; (F) PCP + rice; (G) PCP + canola; (H) PCP + vetch. Finally, both amended and unamended soils were transferred to 250 mL beakers to provide 7 cm thick soil depths and equilibrated in a greenhouse for 7 days; Milli-Q water was added to create a 2 mm thick water layer covering the soil surface. The daytime temperature was between 30 and 35 °C, the night-time temperature was between 25 and 30 °C; soils were sampled at 30, 60, and 120 days after incubation by sacrificing individual replicates. Four days before sampling, flooded water above the soil surface in three duplicate beakers was allowed to evaporate and then to reach almost soil saturation. A 20 mL syringe with the top cut off was used to take samples from four profiles (0−10, 10−20, 20−30, and 30−50 mm) separately. Evaporated water was refilled with Milli-Q water every 2 days. PCP and Intermediate Analyses. The concentration of PCP in soils was determined by ultrasonic extraction and solid phase enrichment, followed by HPLC analysis.5 Soil samples (2 g, freeze-dry weight) were adjusted to pH 4 with sulfuric acid and extracted in 10 mL of methanol by ultrasonics (60 kHz, 25 °C) for 15 min. After centrifugation at 3000 rpm for 10 min, the supernatants were collected. The soil residues were then extracted twice more, as above. The supernatant extracts from each soil sample were then combined and concentrated to 1 mL by rotary evaporation. The concentrated extracts were then transferred to an SPE C18 cartridge (6 mL, 500 mg) with 10 mL of Milli-Q water followed by elution with 10 mL of methanol and concentrated to 2 mL under N2. Finally, each sample was filtered through a 0.22 μm Millipore membrane (ANPEL, 13 mm diameter) prior to analysis. Details of the HPLC analysis were described in our previous study.5 All measurements were in triplicates with a variation generally 99.9% purity) was obtained from Merck KGaAb (Darmstadt, Germany). The other analytical grade chemicals were obtained from Sinopharm Chemical Reagent Co., Ltd., Shanghai, China. Electron Acceptors and Donors. Na2SO4 and NaNO3 were used to supply SO42− and NO3− as electron acceptors. The concentrations of Na2SO4 and NaNO3 were 2.8 and 1.7 g kg−1 soil. The concentrations adopted were those normally used to create SO42− and NO3− reducing conditions in soil−water systems.5,25 Crop residues were used to supply electron donors. They were rice straw (Oryza sativa L.), wheat straw (Triticum aestivum L.), canola residue (Brassica chinensis L.), and Chinese milk vetch (Astragalus sinicus L.). They were collected from field sites, oven-dried at 65 °C for 24 h, and then ground to rice > wheat > canola (Figure 2A). The spatial variations of DOC in the soil profiles showed the same trend during the entire incubation. The main trend was that the DOC increased with increasing soil depth during the entire incubation period. In the crop residue treatments, DOC decreased sharply from 30 to 60 days and maintained a slow decline until the end of incubation. However, in the control and electron acceptor treatments, the DOC concentration showed a slow decline from 30 to 60 days and remained constant by the end of incubation at 120 days, especially in the deep layers. The vertical distribution of SO42− increased with increasing soil depth during 30 days of incubation, but the trend was opposite by the end of incubation at 120 days (Figure 2B) except for the SO42− treatment (Figure 2Bc). The addition of NO3− inhibited the SO42− reduction compared to the control treatment. In contrast, the dissipation of SO42− markedly increased with the
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RESULTS PCP Dissipation in Soil Profiles. The dissipation of PCP in soil profiles is shown in Figure 1. Changes in PCP concentrations varied widely following the addition of electron donors and acceptors but showed a general trend in the initial 30 days of incubation, in which PCP decreased with increasing soil depth in all treatments. However, electron donors and acceptors showed opposite influences on the PCP dissipation. The addition of NO3− and SO42− significantly (p < 0.05) inhibited the dissipation of PCP in the deep layer (20−50 mm) with increasing incubation time (Figure 1b,c), whereas the crop residues (Figure 1d−g) enhanced PCP dissipation in all soil profiles. By the end of the incubation, the PCP concentrations were 0.5−13.3, 6.3−30.0, and 6.0−32.2% for the control, NO3−, and SO42− treatments, respectively. As with all of the crop residue treatments, the PCP concentrations were
