Effects of biochar application on the abundance and ...

Science of the Total Environment 625 (2018) 1218–1224

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Effects of biochar application on the abundance and community composition of denitrifying bacteria in a reclaimed soil from coal mining subsidence area Yuan Liu a, Jirong Zhu a, Chengyu Ye a, Pengfei Zhu a, Qingsong Ba a, Jiayin Pang b, Liangzuo Shu a,⁎ a b

College of Life Sciences, Huaibei Normal University, Anhui Key Laboratory of Resource and Plant Biology, Huaibei 235000, China School of Agriculture and Environment and Institute of Agriculture, University of Western Australia, Crawley, Perth, WA 6009, Australia

H I G H L I G H T S

G R A P H I C A L

A B S T R A C T

• Biochar and N fertilizer increased soil nutrients and wheat yield. • Application of 4% biochar increased abundances of nirK and nirS genes. • Biochar increased the diversity of nirK gene. • Biochar caused a slight change in community structure of nirK or nirS.

a r t i c l e

i n f o

Article history: Received 13 July 2017 Received in revised form 30 December 2017 Accepted 1 January 2018 Available online xxxx Editor: Ajit Sarmah Keywords: Biochar Reclaimed soil Denitrification Soil fertility

a b s t r a c t As a new soil amendment, biochar has become an environmentally friendly material. The application of biochar is one of the most promising management practice to improve soil quality. Using a reclaimed soil from a coal mine subsidence area, the plat soil cultivation experiment in this study investigated the effects of biochar application at varying rates on soil properties, the abundance and composition of soil denitrifier communities. Biochar application significantly increased the crop yield which might be associated with the increased level of cation exchange capacity (CEC), total nitrogen (N), ammonium-N, available phosphorus (P) and potassium (K) in soil. In combination with N fertilizer, the abundance of both nirK and nirS genes significantly increased only at biochar application rate of 4% compared with the nil-biochar treatment. Biochar application significantly increased the community diversity of nirK gene, while not for nirS gene. Redundancy analysis showed that the level of nitrate-N (NO− 3 -N), available P, and pH in soil significantly affected community structure of nirK gene, while the nirS community composition was only affected by soil NO− 3 -N level. Our results indicate that biochar application to the reclaimed soil in coal mine subsidence area could influence the abundance and diversity of soil denitrifiers and improve soil nutrients thus crop yield. © 2018 Elsevier B.V. All rights reserved.

1. Introduction ⁎ Corresponding author. E-mail address: [email protected] (L. Shu).

https://doi.org/10.1016/j.scitotenv.2018.01.003 0048-9697/© 2018 Elsevier B.V. All rights reserved.

Coal is an important source of the energy worldwide, and many areas in China have subsided due to coal mining. The current methods

Y. Liu et al. / Science of the Total Environment 625 (2018) 1218–1224

for land reclamation in coal mine subsidence area such as deep-digging, shallow-filling and grouting (Haynes, 2009), leads to the damaged structure of reclaimed soil. Often, the soil organic matter content is quite low, resulting in extremely poor soil fertility. There are many limitations for vegetation on these sites such as high pH, deficiency of nitrogen (N) and phosphorus (P), high levels of soluble salts and heavy metals, and low levels of organic matter and microbial activity limiting nutrient turnover (Adriano et al., 1980; Belyaeva and Haynes, 2012; Asokan et al., 2005). Therefore long-term input of fertilization and organic matter is required to improve soil fertility of reclaimed soil thus crop yield (Haynes, 2009). However, the buildup of soil nutrients due to over-fertilization would result in the decreased fertilizer use efficiency accompanied with the increased risk of environment pollution. In recent years, incorporation of biochar into agricultural soil and mine wastes has become a hot research topic. Biochar is an aromatized solid material with relatively large pore structures and surface area after pyrolysis under complete or incomplete anoxic conditions (Lehmann, 2007). As an emerging soil amendment, biochar application into soil not only improves soil fertility, but also affects the transformation of N which provides nutrients and habitats for soil microorganisms and reduces N leaching (Lehmann et al., 2011; Gul et al., 2015). Nitrogen level and its turnover in soil are important components of soil fertility. Denitrification, an important component of nitrogen (N) cycling in soil ecosystems, is not only the main route for N-loss in soils but also a significant source of greenhouse gas, nitrous oxide (N2O) (Zhu et al., 2013). Soil denitrification is the reduction of nitrate into NO, N2O, and N2, with the nitrite reduction to NO as the rate-limiting step which depends on the availability of the enzyme nitrite reductase (Nir) (Gregory et al., 2003). Many studies have used nir as the major molecular marker of denitrifiers. Nir has two different morphological structures, namely a copper-based reductase and the heme cd1 based reductase (cd1-nir), encoded by nirK and nirS genes, respectively (Chen et al., 2014). The nirK gene in bacteria is highly diverse with great variation in molecular weight while bacterial nirS genes is mainly found in Pseudomonas with similar molecular weights (Enwall et al., 2005). Studies demonstrate that compared with nirS gene, the expression of nirK gene is more sensitive to environmental changes such as fertilizer types and concentrations, crop species, soil pH, soil water content and others (Braker et al., 2010; Djigal et al., 2010; Szukics et al., 2010). All those factors would in turn change the abundance and diversity of denitrifying bacteria. Lehmann et al. (2006) suggested that biochar application inhibited the growth of denitrifying bacteria, leading to reduced denitrification rate compared with that of the control treatment. Chen et al. (2017) also showed that the addition of biochar not only inhibited the abundance of the nirK gene significantly, but also affected the community composition in paddy soils. However, Ducey et al. (2013) and Anderson et al. (2011) indicated that the abundance of organisms involved in denitrification processes was increased in biochar amended soils. These contradictory results highlight the need for further investigation on biochar's biotic effect and its interaction with soil microorganism. So far, the effects of biochar application to reclaimed soil in coal mining subsidence area on the abundance of denitrifiers and the community composition in remain largely unknown. Coal mine subsidence area in Huaibei, an important coal mining base in China, has reached 157.86 km2 and is predicted to be over 300 km2 by 2020 (Zhu, 2009). In the present study, varying rates of biochar were supplied to the reclaimed soil in pots to investigate the changes in soil properties, crop yield and denitrifier communities. The objectives of this study were to evaluate: 1) if biochar amendment could increase crop yield and soil nutrients; 2) how biochar amendment influences the abundance and community structure of denitrifying bacteria in reclaimed soil of the coal mining subsidence area.

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2. Materials and methods 2.1. Site description and experiment setup The soil was collected from the newly reclaimed coal mine subsidence area (34°04′N, 116°93′E) in Shitai Town, Huaibei, Anhui province, China. The area has a temperate and monsoonal climate with an annual mean precipitation of 830 mm, temperature of 14.8 °C, and frost-free period of 202 days. This reclamation area followed the deep-digging and shallow-filling methods to flatten the soil. The top 20 cm newly reclaimed soil was collected in October 2012, air dried, then passed through a 2-mm mesh sieve. The biochar used for the experiment was purchased from Henan Sanli New Energy Co., Ltd., which was obtained by anaerobic pyrolysis under 400–550 °C. Basic physicochemical properties of the collected soils and biochar are shown in Table 1. The study was composed of five treatments, i.e., five different levels of biochar, here referred to as N + 0% biochar (C0N1), N + 0.5% biochar (C1N1), N + 1% biochar (C2N1); N + 2% biochar treatment (C3N1), and N + 4% biochar (C4N1). In addition, another treatment with no additional C and N fertilizer was set up as an additional control (C0N0). Prior to filling the pots, the fertilizers (N at a rate of 200 mg N kg−1 as ammonium sulfate, P and K as KH2PO4 and K2SO4) and biochar were mixed thoroughly with the reclaimed soil. Each pot was filled with 22.5 kg soil. During the experiment, a soil moisture sensor (SWP-100, China) was inserted in each pot, and the soil moisture was monitored and kept at 65–85% of its field capacity by regular watering. Forty-five seeds of wheat (Triticum aestivum L.) cultivar Jimai 22 were planted in each pot and thinned to 25 plants per pot when seedlings were at five-leaf stage. The plant soil cultivation experiment was carried out in an open air from November 2012 to June 2013. The experiment had four replicates, with each replicate having four pots. 2.2. Crop yield and Total biomass measurement Wheat plants were harvested at physiological maturity. Grains were separated and grain weigh (yield) recorded after drying under 105 °C for 30 min and kept at 70 °C until constant weight were reached. Shoots were cut at ground level and roots were carefully separated from soil. In each plot, total biomass was calculated as the shoot biomass plus root biomass. After oven drying at 70 °C to constant weight (about 48 h), dry weights were determined. 2.3. Analysis of soil physical-chemical properties At final harvest, soil samples were taken to measure the concentra− tion of NH+ 4 -N, NO3 -N, total nitrogen (TN), soil pH, cation exchange capacity (CEC), available phosphorus (P) and potassium (K), and the abundance and community structure of microorganisms. Soil subsamples were taken and kept under − 80 °C for DNA extraction later. Soil − NH+ 4 -N and NO3 -N were determined after extracted with 1 M KCl solution at a soil:water ratio of 1:5 at 25 °C on a Continuous Flow Chemical Analytical System (AA3, Germany) (Zhang et al., 2014). Soil pH was measured at soil:water ratio of 1:2.5; and total nitrogen was determined using the semi-micro Kjeldahl method. The available P was extracted with 0.5 M NaHCO3 and measured with a colorimetric method. Available K was extracted with 1.0 M ammonium acetate solution (pH 7.0) and determined with flame photometer (BWB-XP, England). 2.4. DNA extraction and real time PCR assay Total DNA in soil was extracted from 0.35 g soil with a PowerSoil™ DNA Isolation Kit (MoBio, Carlsbad, CA, USA) following the manufacturer's instructions. DNA fragment size was visualized in 0.8% agarose gel and its size determined with a Nanodrop spectrophotometer (Thermo Scientific, DA, USA).

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Table 1 Basic physicochemical properties of the reclaimed soil and biochar. Soil type

pH

CEC (cmol·kg−1)

Organic matter (g·kg−1)

Total nitrogen (g·kg−1)

Available phosphorus (g·kg−1)

NH+ 4 -N (mg·kg−1)

NO− 3 -N (mg·kg−1)

Reclaimed soil Biochar

8.42 9.31

18.31 14.51

4.27 737

0.29 10.13

0.53 1.58

12.53 8.23

32.76 –

Real-time PCR was performed in a 7500 PCR system via fluorometric monitoring with SYBR Green 1 dye. The primer pairs nirK876/nirK1040 (Henry et al., 2004) and nirS4F/nirS6R (Braker et al., 1998) were used to quantify denitrifier nirK and nirS genes, respectively. Each reaction was performed in a 25 μL volume mixture containing 15 ng DNA, 1 μL10 μM forward and reverse primers, and 12.5 μL SYBR premix EX Taq ™. Melting curve analysis of PCR products was conducted following each assay to confirm that specific amplification was not from primer-dimers or other artifacts. Standard curves were generated using a 10-fold dilution of plasmid DNA, from 103 to 109 copies of the template. PCR efficiency obtained were between 98% and 107%, with R2 values over 0.98.

2.5. T-RFLP analysis of nirK and nirS communities Terminal-restriction fragment length polymorphism (T-RFLP) was used in analyzing nirK and nirS community structure. Briefly, nirK and nirS genes were amplified by PCR using the primer pairs nirK1F/nirK5R and nirS4F/nirS6R (Avrahami et al., 2003) but with the 5′ ends of both the nirK1F and nirS4F primers labeled with 6-carboxyfluorescein (6FAM). Each reaction was performed in a 50 μL mixture containing 1 μL DNA template, 1 μL10 μM forward and reverse primers, and 25 μL Go Taq Green Master Mix. PCR products were detected using 1.5% agarose gel, and directly purified using the PCR purification kit. Purified PCR products were further digested with the restriction enzyme HhaIII (Takara) for nirK genes and HhaI (Takara) for nirS gene. Digests were incubated at 37 °C for 5 h and subsequently inactivated by heat denaturation at 65 °C for 20 min. Fragment analysis was undertaken by capillary electrophoresis (ABI3100 Genetic Analyzer), using a GeneScanROX-labeled GS500 internal size standard. T-RFLP patterns were produced using the GeneMapper software (Applied Biosystems), and peaks between 50 and 550 bp were selected to avoid T-RFs caused by primer-dimers. The relative abundance of a true T-RF within a given T-RFLP pattern was generated as a ratio of the respective peak heights. The peaks with heights b2% of the total peak height were not included for further analyses. The Shannon diversity index (H′) was used to calculate the diversity of nirK and nirS-containing communities based on the following equations:

Shannon index ¼ −

X ðni=NÞ ln ðni=NÞ

where, ni denotes relative abundance of the ithT-RF, i refers to the numbering of each T-RF in T-RFLP spetrum, and N is the total sum of relative abundances of selected T-RF for all samples in the T-RFLP spectrum.

2.6. Statistical analysis Statistical analysis was performed using SPSS 20.0. One-way analysis of variance (ANOVA) followed by Duncan's test was used to test significant differences among the treatments with a probability defined at p b 0.05. Redundancy analysis (RDA) with Monte Carlo permutation's test (499 permutations) was used to evaluate the relationship between nirK and nirS community structures and soil properties computed in Canoco 5.0 software.

3. Results 3.1. Effects of biochar application on soil properties and nutrient uptake Nitrogen fertilizer without biochar treatment (C0N1) significantly reduced soil pH to 8.36 compared with that in the reclaimed soil (8.48). The application of biochar in combination with N fertilizer further decreased soil pH ranging from 8.16–8.27 in biochar application treatments (Table 2). Application of N fertilizer alone (C0N1) significantly increased the soil concentration of NO− 3 -N, and TN, but no significant effect on soil cation exchange capacity (CEC) and NH+ 4 -N. Compared with the C0N1, biochar in combination with N fertilizer significantly increased soil CEC, NH+ 4 -N, and TN, with the increase greater at the higher rate of biochar application. However, biochar application had no significant effect on the soil NO− 3 -N concentration in comparison with the C0N1 treatment (Table 2). The application of N fertilizer alone had no effects on the available P or K concentration in soil compared with C0N0. The application of biochar slightly increased the soil available P only at high rates (C3N1 and C4N1), compared with C0N1. Compared with C0N1, soil available K concentration significantly increased by 11.0%, 25.6%, 36.9%, and 90.8% in C1N1, C2N1, C3N1 and C4N1, respectively (Table 2). Total nutrient uptake of N, P and K by the wheat crop increased overall with biochar and N fertilizer application (Table S1). In this study, total K uptake was significantly increased with increasing biochar application. Compared with C0N1, biochar application significantly increased total K uptake by 30.7% for C2N1, 26.5% for C3N1 and 53.5% for C4N1. However, biochar application had no significant effect on total uptake of N or P when compared with C0N1.

3.2. Effects of biochar application on crop yield and Total biomass Biochar and N fertilizer affected crop yield remarkably (Table 2). Applying N fertilizer alone (C0N1) significantly increased crop yield by 192.1%. Compared with the C0N1 treatment, application of biochar in combinatiion with N fertilizer significanly increased crop yield by 26.3% for C2N1, 30.5% for C3N1 and 53.4% for C4N1. Total biomass were significantly increased by 76.8% in C0N1 treatment compared with that in C0N0. However, biochar application had no effect on total biomass compared with C0N1.

3.3. Effects of biochar application on the abundance of nirK and nirS genes The abundance of nirK gene, ranging from 1.29 × 107 to 4.14 × 107 copies·g−1 dry soil weight in different treatments, was greater than that of nirS gene ranging from 4.75 × 106 to 1.42 × 107 copies g−1 dry soil weight (Fig. 1). In response to the application of biochar and N fertilizer, the abundance of nirK gene followed a similar trend to nirS gene. Biochar and N fertilizer significantly increased the abundance of nirK and nirS genes compared with that in C0N0 (Fig. 1). The application of N fertilizer alone increased the abundance by 100.8% for nirK and 51.2% for nirS, respectively compared with C0N0. Compared with that in the C0N1 treatment, biochar had little influence on the abundance of nirK or nirS gene, except when biochar application was at a rate of


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Table 2 Effects of biochar addition to the reclaimed soil on soil basic properties, crop yield and total biomass of wheat plants. Different lowercase characters indicate significant difference (p b 0.05) between treatments.

C0N0 C0N1 C1N1 C2N1 C3N1 C4N1

pH

CEC (cmol·kg−1)

NH+ 4 -N (mg·kg−1)

NO− 3 -N (mg·kg−1)

TN (g·kg−1)

Available phosphorus (mg·kg−1)

Available potassium (mg·kg−1)

Grain yield (g·pot−1)

Total biomass (g·pot−1)

8.48 ± 0.04a 8.36 ± 0.04b 8.16 ± 0.03d 8.16 ± 0.03d 8.27 ± 0.05c 8.33 ± 0.03bc

20.88 ± 0.65c 21.15 ± 0.63c 22.24 ± 0.41b 23.45 ± 0.45b 24.71 ± 0.72ab 25.77 ± 0.35a

9.95 ± 0.97d 11.74 ± 1.98 cd 13.45 ± 0.62c 17.87 ± 0.67b 19.35 ± 1.92ab 20.49 ± 1.65a

13.66 ± 0.58b 33.96 ± 2.24a 33.73 ± 1.89a 33.07 ± 1.09a 33.01 ± 0.47a 31.90 ± 0.65a

0.24 ± 0.01f 0.27 ± 0.01e 0.33 ± 0.02d 0.46 ± 0.02c 0.61 ± 0.01b 0.77 ± 0.02a

40.78 ± 0.76b 42.14 ± 0.54b 41.88 ± 0.53b 41.46 ± 1.22b 43.62 ± 0.73a 44.47 ± 1.12a

246.05 ± 3.44e 236.43 ± 6.41e 262.49 ± 9.43d 296.96 ± 4.42c 323.62 ± 4.50b 451.19 ± 9.70a

19.10 ± 4.17d 55.80 ± 8.53c 60.70 ± 12.94bc 70.47 ± 13.36b 72.81 ± 7.65b 85.58 ± 7.40a

65.14 ± 8.95b 115.18 ± 11.21a 113.54 ± 10.33a 119.85 ± 8.05a 121.46 ± 7.73a 125.67 ± 9.78a

4.0%, where the abundance of nirK or nirS gene increased by 59.8% and 97.7%, respectively. 3.4. Effects of biochar application on the community composition of nirK and nirS Nitrogen fertilizer alone (C0N1) significantly increased the diversity of nirK and nirS genes compared with the C0N0 (Table 3). When compared with C0N1, biochar application in conjunction with N fertilizer significanly increased the diversity of nirK gene by 15.3% in C2N1, 24.3% in C3N1 and 30.6% in C4N1. In contrast, biochar had no significant effect on the diversity of nirS gene when compared with the C0N1 treatment (Table 3). RDA analysis showed that the first two axes explained about 45.8% and 39.1% of the total variations in nirK and nirS genes, respectively (Fig. 2). No clear differences in nirK or nirS gene communities among the biochar treatments in combination with N fertilizer. In addition, soil pH and the concentration of NO− 3 -N and available P significantly affected community structure of nirK gene, while only soil NO− 3 -N concentration significantly affected the microbial community of nirS gene. 4. Discussion 4.1. Effects of biochar on soil properties and crop yield Biochar has a variety of pore structures and relatively large specific surface area containing many oxygenic functional groups that could adsorb nutrients and water in soil (Liu et al., 2013; Gul and Whalen, 2016). These characteristics are significant especially under the effects of crop root exudates and soil animals, leading to alternation of soil environment to some extent, and then further affecting soil microorganisms. The main effect of returning biochar to fields is a boost in soil fertility and improvement of moisture, porosity, CEC and pH (Lehmann et al., 2006; Tan et al., 2017). This study further demonstrated a consistence of improvement in soil fertility such as CEC, TN, NH+ 4 -N, available P

and K contents. This is in consistent with other studies reported in both upland and flooded soils (Liu et al., 2013; Gul and Whalen, 2016; Zhang et al., 2016a, 2016b). Nitrogen content is a key indicator of soil fertility and an essential element for plant growth. Biochar has been reported to enhance the fixation, mineralization and transformation of organic nitrogen in soils to provide a nitrogen source for soil microbes and increase the soil nitrogen content (Rovira et al., 2009; Tan et al., 2017). This study also showed that the application of biochar to the reclaimed soil further increased available P and K compared with the C0N1 treatment. Similar to the present study, higher available P and K in biochar application treatment was also observed, which might be mainly due to the direct addition of nutrients and reductions in runoff and leaching (Liu et al., 2016; Laird et al., 2010). The total P content of soil is nearly unchanged but the available P content increases significantly after biochar application (Tan et al., 2017). The increasing amount of available P potentially results from the oxidation and combination of Al and Fe in soils with biochar, which releases the constrained P to increase the available P in soils (Deluca et al., 2009). Liu et al. (2017) also suggested that biochar application significantly increased available K in the wheat soils. Biochar that have greater surface area and higher CEC has greater adsorption capacity for ionic forms of P and K rather than leaching out from soil (Liang et al., 2006). In addition, biochar itself could also provide available Ca, Mg, P and K, etc., having the potential to enhance soil fertility (Silber et al., 2010; Liu et al., 2017). This study found that the biochar addition exerted a significant positive effect on wheat yield. It is well-known that biochar application to cropland could increase crop productivity through improving soil fertility (Liu et al., 2013; Gul and Whalen, 2016). In a meta-analysis by Liu et al. (2013), biochar soil amendment increased crop productivity by 11.0% on average over the control. An overall moderate increase in crop yield by 8.4% was visible for the 358 pairs of data reported, while the responses varied with experimental conditions. Enhancement of crop yield with biochar application has been attributed to soil fertility improvement via enhanced availability of nutrients such as P, K, Ca and Mg in soils (Major et al., 2010; Liu et al., 2013), increased N-use efficiency and N retention (Zhang et al., 2014; Zhang et al., 2016a, 2016b) and increased soil moisture regime (Blackwell et al., 2010; Zhang et al., 2012). The increase of soil nutrients was also exhibited in both upland maize and flooded rice crops (Huang et al., 2013; Zhang et al., 2016a, 2016b). In this study, the greater crop yield might be mainly due to the improvement in soil nutrients such as CEC, TN, NH+ 4 -N, available P and K. Therefore, the application of biochar to reclaimed subsidence Table 3 The Shannon-Wiener index (H′) of nirK and nirS genes in the reclaimed soils as affected by biochar application. Different lowercase characters indicate significant difference (p b 0.05) between treatments.

Fig. 1. Effects of biochar application on the abundance of nirK and nirS genes in the reclaimed soil.

Treatment

nirK (H′)

nirS (H′)

C0N0 C0N1 C1N1 C2N1 C3N1 C4N1

0.88 ± 0.06d 1.11 ± 0.05c 1.24 ± 0.07bc 1.28 ± 0.08b 1.38 ± 0.06b 1.45 ± 0.06a

0.62 ± 0.05c 0.83 ± 0.08ab 0.79 ± 0.05b 0.86 ± 0.06a 0.88 ± 0.05a 0.89 ± 0.06a

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Fig. 2. RDA analysis based on the T-RFLP patterns of nirK (A) and nirS (B) and soil properties. Similar symbols with same color in RDA plot indicate the replicated samples.

area soil could improve soil fertility and crop yield, and be used as a promising practice for the management of reclaimed soil in the coal mine subsidence area. 4.2. Effects of biochar on denitrifier abundance and community structure The present study showed that compared with no nitrogen fertilizer application, the addition of N fertilizer significantly increased the abundance and diversity of denitrifiers, and the effect was greater when in combination with biochar (Fig.1 and Table 3). N fertilizer has been found a key factor affecting microbial community diversity and abundance. The application of N fertilizer may weaken plant-microbial competition for N, thus influence microorganisms by direct soil nutrients input and indirect plant effects. Geisseler and Scow (2014) suggested that mineral fertilizers application had a 15.1% increase in microbial biomass by a meta-analysis. In the present study, greater abundance and diversity of denitrifiers under the combination of biochar and N fertilizer conditions coincide with higher soil properties and crop yield (Table 2). Similarly, Doan et al. (2014) and Tian et al. (2016) observed a higher microbial abundance in combination of biochar and N fertilizer addition compared with only mineral fertilizer in both laboratory incubation and paddy field studies. This may be attributed to increased N use efficiency with the application of biochar (Steiner et al., 2008), thus resulting in a greater fertilization effects compared with N fertilizer only. A growing body of evidence suggests that biochar affects microbial community compositions involved in N transformation processes in soils (Lehmann et al., 2011; Doan et al., 2014). The amendment of biochar into soil affects the diversity, abundance and activity of denitrifier communities which are due to the changes in soil geochemical param+ eters, such as pH, oxygen saturation, nitrogen speciation (NO− 3 /NH4 ) and availability (Anderson et al., 2011; Ducey et al., 2013). Anderson et al. (2011) reported increases in the relative abundances of organisms involved in denitrification in biochar amended soil, and changes in soil aeration and water holding capacity could decrease the prevalence of anaerobic pockets where denitrification processes are more likely to occur (Yanai et al., 2007). Ducey et al. (2013) also indicated that the abundance of genes associated with denitrification was increased in biochar amended soils after 6 months. In the present study, we found that in test reclaimed soil, application of biochar increased the abundance of nirK and nirS genes and the stimulation effect increased with the level of biochar application (Fig. 1). When considering

denitrification processes, the increase in abundances of both nirK and nirS indicates enhanced nitrate reduction, which could result less avail− able N for the plant. Biochar could adsorb and fix NH+ 4 -N and NO3 -N in − soil, facilitate transformation of NH+ 4 -N to NO3 -N, and provide more substrates for denitrifiers and denitrification process, thus affecting their community composition (Harter et al., 2016). In a wheat mesocosm experiment, Liu et al. (2017) suggested that biochar significantly increased the abundance of denitrifier gene because the biochar's liming effect resulted an optimum neutral pH level for the growth of a wide variety of denitrifiers. This may be not the cause the present study because no significant increase in soil NO− 3 -N or pH was observed between and biochar treatments (Table 2). However, Liu et al. (2014a, 2014b) suggested that increased labile organic C originating from amended biochar increase denitrification. Hamer et al. (2004) also indicated that biochar may promote decomposition of soil labile C compounds by providing more surface for growth of microorganism. Nonetheless, it is still a challenge to generalize biochar stimulation mechanisms because of various biochar characteristics and hence biochar-soil interactions. Our results showed little change in the nirK or nirS community structure after biochar addition,but a significant increase in the diversity was observed (Fig. 2 and Table 3). Studies have documented that soil denitrifiers are sensitive to environmental changes and responses of different denitrifying genotypes may also differ in response to the environmental factors (Liu et al., 2010; Chen et al., 2010). Changes in soil properties induced by biochar addition could indirectly affect microbial community (Lehmann et al., 2011). In this study, RDA results revealed that soil NO− 3 -N, pH, and available P were the most significant factors influencing denitrifier's community compositions. As the main substrate for denitrification, NO− 3 -N could be a key factor determining the community composition of denitrifiers. Previous studies have documented that soil pH is a key factor to determine the structure of denitrifier communities in selecting different functional groups (Chen et al., 2010; Palmer et al., 2010). ŠImek and Cooper (2002) also found that pH levels could change denitrifier communities by affecting the ability of the microorganisms to utilize the available organic carbon. This study suggested a potential role of biochar in enhancing soil fertility, crop yield as well as modifying denitrifier communities in reclaimed soil from coal mining subsidence area. Therefore, great care should be taken when using biochar as soil amendment such that its use will improve not only the soil physi-chemical properties, but its microbial communities as well. These microbial communities, reflected in


part by the community structure and abundance, will play a significant role in soil N cycling processes. However, the link of these biochar amendment induced changes to soil nutrient cycling processes remained unclear. 5. Conclusion This study indicated that biochar application could significantly improve soil fertility and alter denitrifier communities in the reclaimed soil from a coal mine subsidence area, and these effects were biochar application rate dependent. The biochar and N fertilizer application significantly increased crop yield which might be associated with the improved soil nutrients such as soil CEC, NH+ 4 -N, TN, available P and K contents. The abundance of nirK and nirS genes significantly increased only at biochar application rate of 4% compared with the nil-biochar treatment. The community composition of nirK gene was mainly affected by the changes in soil NO− 3 -N, pH and available P contents. These findings suggest that adding biochar to reclaimed soil from coal mining subsidence with N fertilizer can improve crop yield and soil fertility with altering the abundance and community structure of denitrifiers. Finally, they support the concept that biochar amendment to agriculture could significantly improve crop yield and soil fertility. Supplementary data to this article can be found online at https://doi. org/10.1016/j.scitotenv.2018.01.003. Acknowledgements This work was supported by the Innovation Team of Scientific Research Platform of Anhui Province, China (KJ2015TD001), National Natural Science Foundation of China (31572202, 41501304), and the Research Foundation for Overseas Returnees in College under the Ministry of Education of China. References Adriano, D.C., Page, A.L., Elseewi, A.A., Chang, A.C., Straughan, I., 1980. Utilization and disposal of fly ash and other coal residues in terrestrial ecosystems: a review. J. Environ. Qual. 9, 333–344. Anderson, C.R., Condron, L.M., Clough, T.J., Fiers, M., Stewart, A., Hill, R.A., Sherlock, R.R., 2011. Biochar induced soil microbial community change: implications for biogeochemical cycling of carbon, nitrogen and phosphorus. Pedobiologia 54, 309–320. Asokan, P., Saxena, M., Asolekar, S.R., 2005. Coal combustion residues- environmental implications and recycling potentials. Resour. Conserv. Recycl. 43, 239–262. Avrahami, S., Liesack, W., Conrad, R., 2003. Effects of temperature and fertilizer on activity and community structure of soil ammonia oxidizers. Environ. Microbiol. 5, 691–705. Belyaeva, O.N., Haynes, R.J., 2012. Comparison of the effects of conventional organic amendments and biochar on the chemical, physical and microbial properties of coal fly ash as a plant growth medium. Environ. Earth Sci. 66, 1987–1997. Blackwell, P., Krull, E., Butler, G., Herbert, A., Solaiman, Z., 2010. Effect of banded biochar on dryland wheat production and fertilizer use in south-western Australia: an agronomic and economic perspective. Aust. J. Soil Res. 48, 531–545. Braker, G., Fesefeldt, A., Witzel, K.P., 1998. Development of PCR primer systems for amplification of nitrite reductase genes (nirK and nirS) to detect denitrifying bacteria in environmental samples. Appl. Environ. Microbiol. 64, 3769–3775. Braker, G., Schwarz, J., Conrad, R., 2010. Influence of temperature on the composition and activity of denitrifying soil communities. FEMS Microbiol. Ecol. 73, 134–148. Chen, Z., Luo, X., Hu, R., Wu, M., Wu, J., Wei, W., 2010. Impact of long-term fertilization on the composition of denitrifier communities based on nitrite reductase analyses in a paddy soil. Microb. Ecol. 60, 850–861. Chen, Y., Zhou, W., Li, Y., Zhang, J., Zeng, G., Huang, A., Huang, J., 2014. Nitrite reductase genes as functional markers to investigate diversity of denitrifying bacteria during agricultural waste composting. Appl. Microbiol. Biotechnol. 98, 4233. Chen, J., Li, S., Liang, C., Xu, Q., Li, Y., Qin, H., Fuhrmann, J.J., 2017. Response of microbial community structure and function to short-term biochar amendment in an intensively managed bamboo (Phyllostachys praecox) plantation soil: effect of particle size and addition rate. Sci. Total Environ. 574, 24–33. Deluca, T., Mackenzie, M., Gundale, M., 2009. Biochar effects on soil nutrient transformation. In: Lehmann, J., Joseph, S. (Eds.), Biochar for Environmental Management: Science and Technology. Earthscan, pp. 251–265. Djigal, D., Baudoin, E., Philippot, L., Brauman, A., Villenave, C., 2010. Shifts in size, genetic structure and activity of the soil denitrifier community by nematode grazing. Eur. J. Soil Biol. 46, 112–118.

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