Appl Microbiol Biotechnol (2014) 98:263–272 DOI 10.1007/s00253-013-5311-1
APPLIED GENETICS AND MOLECULAR BIOTECHNOLOGY
Effects of roxithromycin on ammonia-oxidizing bacteria and nitrite-oxidizing bacteria in the rhizosphere of wheat Binbin Yu & Xin Wang & Shuai Yu & Qiang Li & Qixing Zhou
Received: 13 July 2013 / Revised: 1 October 2013 / Accepted: 2 October 2013 / Published online: 23 October 2013 # Springer-Verlag Berlin Heidelberg 2013
Abstract In a pot-cultural experiment, the impact of the antibiotic roxithromycin (ROX) addition was assessed on the diversities of microbial structure and function communities, especially involved in ammonia and nitrite oxidation in wheat rhizosphere soil with and without the addition of earthworms. The abundances of ammoniaoxidizing bacteria (AOB), nitrite-oxidizing bacteria (NOB), and total bacteria were surveyed by the quantitative PCR. The quantities of total bacteria, AOB, and NOB with earthworms were higher than those without earthworms because of the synergistic effect. ROX inhibited the growth of AOB in all treatments, although the quantities of AOB were in a light increase in medium and heavy polluted treatments compared with that in the light polluted treatments. Different from AOB, the quantities of NOB were lowest in light polluted treatments, but the quantities of NOB were rapidly increased in medium and heavy polluted treatments compared with that in the control. These results indicated that the application of ROX principally had a negative effect on nitrification performance by affecting the abundances and relative ratios of both AOB and NOB in soil communities, which affected the N cycle in an agricultural ecosystem. According to the metabolic diversities evaluated by the biologic assay, the tendency of metabolic diversities was quite contrary to the quantities of NOB in all treatments and showed B. Yu : X. Wang : S. Yu : Q. Zhou (*) MOE Key Laboratory of Pollution Processes and Environmental Criteria, College of Environmental Science and Engineering, Nankai University, Tianjin 300071, China e-mail: [email protected] Q. Li MOE Key Laboratory of Molecular Microbiology and Technology, College of Life Science, Nankai University, Tianjin 300071, China
the contrast growing relation of autotrophic and heterotrophic bacteria under ROX pollution pressure in agricultural ecosystems. Keywords Roxithromycin . AOB . NOB . Heterotrophs . Earthworm
Introduction For several decades, to treat or prevent microbial infections and promote the growth of animals in livestock and aquaculture production, antibiotics have been widely used in human and veterinary medicine (Hirsch et al. 1999; Martinez 2009). The annual usage of antibiotics has been estimated to be ranged from 100,000 to 200,000 tons in the world (Kümmerer 2009), while more than 25,000 tons were used in China each year (Gao et al. 2012; Jiang et al. 2011). A large number of these pharmaceuticals or their primary metabolites are excreted rapidly after administration by human and animals. Thus, large fractions of antibiotics and their metabolites are transformed into soils through manure fertilization, sewers, sewage treatment plants, and sewage sludge, which could affect the normal life activities of plants, animals, and microorganisms, and eventually have effects on human health through food chain. At present, most of environmental researches about antibiotics pollution mainly focus on the mechanisms of pollution and degradation (Wetzstein et al. 1999). The effects of antibiotics on environmental microbial ecology are less investigated (Martinez 2009). Antibiotic is an important type of antimicrobial agents that can directly kill or inhibit certain microorganisms in environmental media such as soil, water, and sediments, and therefore change the microbial community structure (Haller et al. 2002; Hammesfahr et al. 2008; Yang et al.
264
2009), function (Kong et al. 2006; Schauss et al. 2009), and activities (Boleas et al. 2005; Kotzerke et al. 2008; Thiele-Bruhn and Beck 2005). Antibiotics discharged into soil may (1) affect the reproduction, metabolism, and activity of soil microorganisms; (2) change community structure and biodiversity; and (3) affect soil biogeochemical processes of an element and the selfpurification capacity. Nitrogen cycle is essential to energy conversion and balance in an ecosystem and agricultural production activities. Nitrification plays a vital role in this nitrogen cycle, where ammonium (NH 4 + ) is converted into nitrite (NO2−) and nitrate (NO3−) in soil during the decomposition process (Norton et al. 2002; Yamamoto et al. 2010). In fact, there are two reactions: NH4+ is firstly oxidized to NO2− by ammonia-oxidizing bacteria (AOB), and then NO2− is further oxidized to NO3− by nitrite-oxidizing bacteria (NOB) (Kowalchuk and Stephen 2001). AOB controls nitrification in the firststage reaction, which is one of the key reactions of nitrate formed in soil N circulation; and NOB is very important to convert NO2− into NO3− (De Boer et al. 1995; Nicol et al. 2008). Roxithromycin (ROX), as one of emerging semisynthetic macrolide antibiotics that are widely used in respiratory infection, was founded in wastewater (Segura et al. 2009), surface water (Gao et al. 2012; Hirsch et al. 1998; Tang et al. 2009), and sediments (Gao et al. 2012; Tang et al. 2009). ROX reaches the soil via watering and manuring. Although the concentration of ROX was lower than other antibiotics in the environment, it cannot be easily degraded in soil (Schlüsener and Bester 2006). Earthworms are ubiquitous soil invertebrates. They ingest large amounts of mineral soil and organic materials (Pedersen and Hendriksen 1993), which result in great influences of the physical and chemical composition of soil (Furlong et al. 2002). Earthworms play a synergistic effect on microorganisms in the environment filled with biodegradable organic matter, carbon (C), nitrogen (N), phosphorus (P), potassium (K), and other nutrients into a form more conducive to plant uptake, enhance the microbial diversity, and improve crop yields (Edwards and Fletcher 1988; Pedersen and Hendriksen 1993). When antibiotics discharged into an agricultural ecosystem, they can affect and change microorganism community in soil. Although there are some studies about the effects of antibiotics on microbial communities in soil, limited studies were performed on nondegraded and small-dose antibiotics up to date. Thus, we want to investigate that ROX has effects on the abundances of nitrifying bacteria whether or not and how it affects. Therefore, in this study, we prepared pot-culture experiments to
Appl Microbiol Biotechnol (2014) 98:263–272
investigate the variation in abundances and quantities of AOB and NOB, nitrification performance, as well as the community function under ROX pollution pressure with and without the addition of earthworms.
Materials and methods Soil preparation and treatments The soil was collected from the surface layer (0–10 cm) of unpolluted agricultural fields in the Jixian County, Tianjin (39°56′07 N, 117°22′37 E). These samples were air-dried and screened through a 4-mm mesh before used in the potculture experiments. Basic physicochemical properties of the soil were analyzed according to the routine analytical methods of agricultural chemistry in soil. The pH is 7.2, organic matter content is 64.77 g kg−1, total N content is 1.48 g kg−1, and total P content is 1.48 %. Sixty kilograms soil samples were placed in a plastic basin after mixing with the appropriate amount of ROX (Sigma, USA). According to the concentration of ROX in sediment environment (Gao et al. 2012; Tang et al. 2009; Zhou et al. 2011), six samples with different ROX contents were applied, including CK (the control, no ROX added) and three of ROX content levels represent for light (0.2 and 0.5 mg kg−1), medium (1.0 and 2.0 mg kg−1), and heavy (10 mg kg−1) contaminations. The soil was watered to a humidity of 30– 40 % and then left outdoors to equilibrate under a waterproof tarpaulin for 3 weeks, which was long enough to allow the natural equilibration of various sorption mechanisms in soil. Then, after mixing thoroughly, soil of each level was placed in separate plastic pots, with 2.5 kg of soil added in each port (20 cm in diameter and 15 cm in height). Plant culture Seeds of wheat (Triticum aestivum L.) were surface-sterilized in 3 % (v/v) H2O2 for 10 min and rinsed with deionized water by several times before germination. Then, they were placed on moistened filter paper and incubated in the dark at 25±2 °C in a culturing box. When the plumules of seeds were seen, 50 seeds were placed in each plot. Earthworms (Eisenia foetida) were washed with deionized water, then placed in a plastic pot for 2 days to put purge. After seeds were planted, earthworms were added or not in parallel pots at each ROX content at the same day. The plants were cultivated under greenhouse without any fertilization. After growing, rhizosphere soils, wheat plants, and earthworms were sampled at 7, 21, 35, 49, 63, 77, 91,105, and 140 days. Soil samples, wheat and earthworms were stored in a freezer at −20 °C before DNA extraction.
Appl Microbiol Biotechnol (2014) 98:263–272
265
DNA extraction from soil samples
Biology eco-assay
Total DNA was extracted from 0.5 g of soil using an E.Z.N.A.TM Soil DNA Kit (Omega Biotek, USA), following the manufacturer's instructions. Successful extracted DNA was determined by 0.8 % (wt/vol) agarose gel electrophoresis. The quantity and purity of extracted DNA were detected by UV spectrophotometry (Bio-RAD, USA) at 260 and 280 nm. Extracted DNA was stored at −20 °C.
The fresh rhizospheric soil sample equivalent to 1.0 g dried soil was dissolved in 10 mL aquae sterilisata, then scrolled for 10 min to mix thoroughly and diluted to 10–3 gradually. One hundred fifty microliters of the diluted soil suspension was inoculated into each well of an Eco Plate (Biolog, USA), which contains 31 individual carbon sources in triplicate and 3 negative control ( without carbon source) in a 96-well plate format. The plates were incubated at 25 °C for 168 h and read under 590 nm (OD590) using a plate reader (Biolog, USA).
Quantification of AOB, NOB, and total bacteria by quantitative PCR
Analytical methods For analysis of community of AOB and NOB, PCR targeting the region of 16S rRNA genes, AOB was performed using beta-proteobacterial AOB-specific primer pair CTO-189f and CTO-654r (CTO-189f: 5'-CTA GCY TTG TAG TTT CAA ACG C-3′ and CTO-654r: 5'-CTA GCY TTG TAG TTT CAA ACG C-3′) (Kowalchuk et al. 1997); furthermore, NOB was performed using Nitrospira NOB-specific primer pair NSR1113f and NSR-1264r ( NSR-1113f: 5′-CCT GCT TTC AGT TGC TAC CG-3′ and NSR-1264r: 5′-GTT TGC AGC GCT TTG TAC CG-3′) (Dionisi et al. 2002). Community analysis of total bacteria was performed in a region from V3 region using primer pair 357f and 518r (357f: 5′-CCT ACG GGA GGC AGC AG-3′ and 518r: 5′-ATT ACC GCG GCT GCT GG-3′) (Muyzer et al. 1993). Quantitative PCR assays were performed to quantify AOB, NOB specific 16S rRNA, and total bacteria genes in triplicate with an iQ 5 Multicolor Real-Time PCR Detection System (BIO-RAD, USA) using the SYBR green IqPCR method. The PCR mixture was prepared in a total volume of 25 μL using a SYBR Premix Ex TaqTM II (Perfect Real Time) (TaKaRa, China). Twenty-five microliter mixture contained the following: 12.5 μL of 2×SYBR GreenIPCR Mix, 0.12– 0.2 μM each primer (0.12 μM for total bacteria 357f and 518r, 0.2 μM for CTO 189f and CTO 654r, NSR 1113f and 1264r), and 1 μL fivefold diluted extracted DNA. All reactions were carried out in 96 PCR plates (Axygen, USA) with transparent films (BIO-RAD, USA). Quantification of AOB, NOB, and total bacteria was carried out in triplicate using a Bio-Rad Q5 Real-Time PCR system under the following thermocycling conditions: 95 °C for 30 s, 40 cycles of 95 °C for 10 s, 58 °C (primers for AOB and total bacteria) or 62 °C (primers for NOB) for 15S, 72 °C for 25 s. The amplification specificities of AOB, NOB, and total bacteria were confirmed by generating melting curves. The standard plasmid DNA were diluted to 1.517×105– 1.517×1010 (total bacteria 16S rRNA), 1.946×105–1.946× 1010 (AOB 16S rRNA), 1.41×105–1.41×1010 copiesμL−1 (NOB 16S rRNA), respectively. The PCR efficiencies were 98.5 % for total bacteria, 89.2 % for AOB, and 105.6 % for NOB, respectively.
All measurements mentioned above were replicated for three times. The data utilized for the analysis were calculated from the median of three paralleled measurements at each harvest time. The treatments of ROX and quantities data of AOB and NOB were analyzed by correlation analysis.
Results Quantities of AOB, NOB, and total bacteria in pot-culture experiments The relationship of environmental variables and ecosystem functioning could be analyzed by the abundances of microorganisms. Under the ROX pressure, the abundances of AOB, NOB, and total bacteria have changed. Meanwhile, earthworms played an important synergistic effect of microorganisms and enhanced microbial diversities. The total bacteria abundances in the group without earthworms were ranged from (2.71±0.45)×108 to (1.49±0.31)×109 copies g−1 dry soil weight, with values 7–22 % lower than those in the group with earthworms [from (2.89±0.28)×108 to (1.92± 0.66)× 109 copies g−1 dry soil weight]. The addition of earthworms increased the abundance of AOB from 8.8 to 37 %, which ranged from (5.84±0.62)×105–(5.20±0.62)× 106 to (6.40±1.06)×105–(8.29±0.86)×106 copies g−1 dry soil weight, while the abundance of NOB increased from (4.13± 0.54)×105–(6.90±1.98)×106 to (4.96±0.83)×105–(8.91± 0.95)×106 copies g−1 dry soil weight, the increase rate was from 16.73 to 22.56 % (Fig. 1). Meanwhile, comparing with the control, the quantities of AOB, NOB, and total bacteria in other treatments were changed evidently (Fig. 1). In two groups without and with earthworms, the quantities of total bacteria in light, medium, and heavy polluted treatments were gradually increased compared to the control, although the quantities of AOB and NOB changed differently. The addition of ROX in light polluted treatments (0.20 and 0.50 mg kg−1) decreased the quantities of AOB by 73–78 % in treatments without
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Appl Microbiol Biotechnol (2014) 98:263–272
quantities of total bacteria, AOB and NOB (copies /g dry soil weight)
quantities of total bacteria, AOB and NOB (copies /g dry soil weight)
quantities of total bacteria, AOB and NOB (copies /g dry soil weight)
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Fig. 1 Abundances and quantities of total bacteria, AOB, and NOB in different treatments. 16S means the total bacteria. 16S, AOB, and NOB present the group without earthworms. 16S+, AOB+, and NOB+ present
the group with earthworms. a–h and i present time profiles of 7, 21, 35, 44, 65, 77, 91, 104 and 140 days
earthworms and 80–85 % with earthworms, while the decrease in NOB quantities was 45–52 % without earthworms and 62–69 % with earthworms. In medium- and heavy-polluted treatments (1.00, 2.00, and 10.00 mg kg−1), the addition of ROX decreased the quantities of AOB by 57, 61, and 66 % in treatments without earthworms and 64, 71, and 76 % with earthworms, in contrast to AOB, the quantities of NOB were increased by 18, 29, and 12 % in treatments without earthworms and 14, 15, and 46 % with earthworms, respectively. Moreover, AOB in the control treatment was significantly higher (p =0.000) than that in other treatments without and with earthworms (Table 1), while NOB in medium and heavy treatments (1.00, 2.00, and
10.00 mg kg−1) were significantly correlated to other treatments (p =0.000; Table 2). Ratios of AOB and NOB in different treatments It is not well established whether there is a ratio of AOB to NOB responsible to the good performance of nitrification, yet the ratio of AOB/NOB is 2.00 through theoretical methods (Hagopian and Riley 1998; Hooper et al. 1997). The reason is that the energy generated from ammonia oxidization by AOB is higher than that produced from nitrite oxidization by NOB. Then, some other studies indicated that a ratio of AOB/NOB of 2.00–3.50 was appropriate related to the inherent high
Appl Microbiol Biotechnol (2014) 98:263–272 Table 1 Statistical evaluation of the quantities of AOB and AOB+ in different treatments by the correlation analysis
AOB presents the group without earthworms. AOB+ presents the group with earthworms.
Group
267 Concentration (mg kg−1)
0 AOB
0
1.000
AOB+
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0.000 0.000 0.000 0.000 0.000 1.000 0.000 0.000 0.000 0.000 0.000
a
The p values show the impact of the ROX treatments on the quantities of AOB and AOB+. Italicized values indicate significant effects (p
APPLIED GENETICS AND MOLECULAR BIOTECHNOLOGY
Effects of roxithromycin on ammonia-oxidizing bacteria and nitrite-oxidizing bacteria in the rhizosphere of wheat Binbin Yu & Xin Wang & Shuai Yu & Qiang Li & Qixing Zhou
Received: 13 July 2013 / Revised: 1 October 2013 / Accepted: 2 October 2013 / Published online: 23 October 2013 # Springer-Verlag Berlin Heidelberg 2013
Abstract In a pot-cultural experiment, the impact of the antibiotic roxithromycin (ROX) addition was assessed on the diversities of microbial structure and function communities, especially involved in ammonia and nitrite oxidation in wheat rhizosphere soil with and without the addition of earthworms. The abundances of ammoniaoxidizing bacteria (AOB), nitrite-oxidizing bacteria (NOB), and total bacteria were surveyed by the quantitative PCR. The quantities of total bacteria, AOB, and NOB with earthworms were higher than those without earthworms because of the synergistic effect. ROX inhibited the growth of AOB in all treatments, although the quantities of AOB were in a light increase in medium and heavy polluted treatments compared with that in the light polluted treatments. Different from AOB, the quantities of NOB were lowest in light polluted treatments, but the quantities of NOB were rapidly increased in medium and heavy polluted treatments compared with that in the control. These results indicated that the application of ROX principally had a negative effect on nitrification performance by affecting the abundances and relative ratios of both AOB and NOB in soil communities, which affected the N cycle in an agricultural ecosystem. According to the metabolic diversities evaluated by the biologic assay, the tendency of metabolic diversities was quite contrary to the quantities of NOB in all treatments and showed B. Yu : X. Wang : S. Yu : Q. Zhou (*) MOE Key Laboratory of Pollution Processes and Environmental Criteria, College of Environmental Science and Engineering, Nankai University, Tianjin 300071, China e-mail: [email protected] Q. Li MOE Key Laboratory of Molecular Microbiology and Technology, College of Life Science, Nankai University, Tianjin 300071, China
the contrast growing relation of autotrophic and heterotrophic bacteria under ROX pollution pressure in agricultural ecosystems. Keywords Roxithromycin . AOB . NOB . Heterotrophs . Earthworm
Introduction For several decades, to treat or prevent microbial infections and promote the growth of animals in livestock and aquaculture production, antibiotics have been widely used in human and veterinary medicine (Hirsch et al. 1999; Martinez 2009). The annual usage of antibiotics has been estimated to be ranged from 100,000 to 200,000 tons in the world (Kümmerer 2009), while more than 25,000 tons were used in China each year (Gao et al. 2012; Jiang et al. 2011). A large number of these pharmaceuticals or their primary metabolites are excreted rapidly after administration by human and animals. Thus, large fractions of antibiotics and their metabolites are transformed into soils through manure fertilization, sewers, sewage treatment plants, and sewage sludge, which could affect the normal life activities of plants, animals, and microorganisms, and eventually have effects on human health through food chain. At present, most of environmental researches about antibiotics pollution mainly focus on the mechanisms of pollution and degradation (Wetzstein et al. 1999). The effects of antibiotics on environmental microbial ecology are less investigated (Martinez 2009). Antibiotic is an important type of antimicrobial agents that can directly kill or inhibit certain microorganisms in environmental media such as soil, water, and sediments, and therefore change the microbial community structure (Haller et al. 2002; Hammesfahr et al. 2008; Yang et al.
264
2009), function (Kong et al. 2006; Schauss et al. 2009), and activities (Boleas et al. 2005; Kotzerke et al. 2008; Thiele-Bruhn and Beck 2005). Antibiotics discharged into soil may (1) affect the reproduction, metabolism, and activity of soil microorganisms; (2) change community structure and biodiversity; and (3) affect soil biogeochemical processes of an element and the selfpurification capacity. Nitrogen cycle is essential to energy conversion and balance in an ecosystem and agricultural production activities. Nitrification plays a vital role in this nitrogen cycle, where ammonium (NH 4 + ) is converted into nitrite (NO2−) and nitrate (NO3−) in soil during the decomposition process (Norton et al. 2002; Yamamoto et al. 2010). In fact, there are two reactions: NH4+ is firstly oxidized to NO2− by ammonia-oxidizing bacteria (AOB), and then NO2− is further oxidized to NO3− by nitrite-oxidizing bacteria (NOB) (Kowalchuk and Stephen 2001). AOB controls nitrification in the firststage reaction, which is one of the key reactions of nitrate formed in soil N circulation; and NOB is very important to convert NO2− into NO3− (De Boer et al. 1995; Nicol et al. 2008). Roxithromycin (ROX), as one of emerging semisynthetic macrolide antibiotics that are widely used in respiratory infection, was founded in wastewater (Segura et al. 2009), surface water (Gao et al. 2012; Hirsch et al. 1998; Tang et al. 2009), and sediments (Gao et al. 2012; Tang et al. 2009). ROX reaches the soil via watering and manuring. Although the concentration of ROX was lower than other antibiotics in the environment, it cannot be easily degraded in soil (Schlüsener and Bester 2006). Earthworms are ubiquitous soil invertebrates. They ingest large amounts of mineral soil and organic materials (Pedersen and Hendriksen 1993), which result in great influences of the physical and chemical composition of soil (Furlong et al. 2002). Earthworms play a synergistic effect on microorganisms in the environment filled with biodegradable organic matter, carbon (C), nitrogen (N), phosphorus (P), potassium (K), and other nutrients into a form more conducive to plant uptake, enhance the microbial diversity, and improve crop yields (Edwards and Fletcher 1988; Pedersen and Hendriksen 1993). When antibiotics discharged into an agricultural ecosystem, they can affect and change microorganism community in soil. Although there are some studies about the effects of antibiotics on microbial communities in soil, limited studies were performed on nondegraded and small-dose antibiotics up to date. Thus, we want to investigate that ROX has effects on the abundances of nitrifying bacteria whether or not and how it affects. Therefore, in this study, we prepared pot-culture experiments to
Appl Microbiol Biotechnol (2014) 98:263–272
investigate the variation in abundances and quantities of AOB and NOB, nitrification performance, as well as the community function under ROX pollution pressure with and without the addition of earthworms.
Materials and methods Soil preparation and treatments The soil was collected from the surface layer (0–10 cm) of unpolluted agricultural fields in the Jixian County, Tianjin (39°56′07 N, 117°22′37 E). These samples were air-dried and screened through a 4-mm mesh before used in the potculture experiments. Basic physicochemical properties of the soil were analyzed according to the routine analytical methods of agricultural chemistry in soil. The pH is 7.2, organic matter content is 64.77 g kg−1, total N content is 1.48 g kg−1, and total P content is 1.48 %. Sixty kilograms soil samples were placed in a plastic basin after mixing with the appropriate amount of ROX (Sigma, USA). According to the concentration of ROX in sediment environment (Gao et al. 2012; Tang et al. 2009; Zhou et al. 2011), six samples with different ROX contents were applied, including CK (the control, no ROX added) and three of ROX content levels represent for light (0.2 and 0.5 mg kg−1), medium (1.0 and 2.0 mg kg−1), and heavy (10 mg kg−1) contaminations. The soil was watered to a humidity of 30– 40 % and then left outdoors to equilibrate under a waterproof tarpaulin for 3 weeks, which was long enough to allow the natural equilibration of various sorption mechanisms in soil. Then, after mixing thoroughly, soil of each level was placed in separate plastic pots, with 2.5 kg of soil added in each port (20 cm in diameter and 15 cm in height). Plant culture Seeds of wheat (Triticum aestivum L.) were surface-sterilized in 3 % (v/v) H2O2 for 10 min and rinsed with deionized water by several times before germination. Then, they were placed on moistened filter paper and incubated in the dark at 25±2 °C in a culturing box. When the plumules of seeds were seen, 50 seeds were placed in each plot. Earthworms (Eisenia foetida) were washed with deionized water, then placed in a plastic pot for 2 days to put purge. After seeds were planted, earthworms were added or not in parallel pots at each ROX content at the same day. The plants were cultivated under greenhouse without any fertilization. After growing, rhizosphere soils, wheat plants, and earthworms were sampled at 7, 21, 35, 49, 63, 77, 91,105, and 140 days. Soil samples, wheat and earthworms were stored in a freezer at −20 °C before DNA extraction.
Appl Microbiol Biotechnol (2014) 98:263–272
265
DNA extraction from soil samples
Biology eco-assay
Total DNA was extracted from 0.5 g of soil using an E.Z.N.A.TM Soil DNA Kit (Omega Biotek, USA), following the manufacturer's instructions. Successful extracted DNA was determined by 0.8 % (wt/vol) agarose gel electrophoresis. The quantity and purity of extracted DNA were detected by UV spectrophotometry (Bio-RAD, USA) at 260 and 280 nm. Extracted DNA was stored at −20 °C.
The fresh rhizospheric soil sample equivalent to 1.0 g dried soil was dissolved in 10 mL aquae sterilisata, then scrolled for 10 min to mix thoroughly and diluted to 10–3 gradually. One hundred fifty microliters of the diluted soil suspension was inoculated into each well of an Eco Plate (Biolog, USA), which contains 31 individual carbon sources in triplicate and 3 negative control ( without carbon source) in a 96-well plate format. The plates were incubated at 25 °C for 168 h and read under 590 nm (OD590) using a plate reader (Biolog, USA).
Quantification of AOB, NOB, and total bacteria by quantitative PCR
Analytical methods For analysis of community of AOB and NOB, PCR targeting the region of 16S rRNA genes, AOB was performed using beta-proteobacterial AOB-specific primer pair CTO-189f and CTO-654r (CTO-189f: 5'-CTA GCY TTG TAG TTT CAA ACG C-3′ and CTO-654r: 5'-CTA GCY TTG TAG TTT CAA ACG C-3′) (Kowalchuk et al. 1997); furthermore, NOB was performed using Nitrospira NOB-specific primer pair NSR1113f and NSR-1264r ( NSR-1113f: 5′-CCT GCT TTC AGT TGC TAC CG-3′ and NSR-1264r: 5′-GTT TGC AGC GCT TTG TAC CG-3′) (Dionisi et al. 2002). Community analysis of total bacteria was performed in a region from V3 region using primer pair 357f and 518r (357f: 5′-CCT ACG GGA GGC AGC AG-3′ and 518r: 5′-ATT ACC GCG GCT GCT GG-3′) (Muyzer et al. 1993). Quantitative PCR assays were performed to quantify AOB, NOB specific 16S rRNA, and total bacteria genes in triplicate with an iQ 5 Multicolor Real-Time PCR Detection System (BIO-RAD, USA) using the SYBR green IqPCR method. The PCR mixture was prepared in a total volume of 25 μL using a SYBR Premix Ex TaqTM II (Perfect Real Time) (TaKaRa, China). Twenty-five microliter mixture contained the following: 12.5 μL of 2×SYBR GreenIPCR Mix, 0.12– 0.2 μM each primer (0.12 μM for total bacteria 357f and 518r, 0.2 μM for CTO 189f and CTO 654r, NSR 1113f and 1264r), and 1 μL fivefold diluted extracted DNA. All reactions were carried out in 96 PCR plates (Axygen, USA) with transparent films (BIO-RAD, USA). Quantification of AOB, NOB, and total bacteria was carried out in triplicate using a Bio-Rad Q5 Real-Time PCR system under the following thermocycling conditions: 95 °C for 30 s, 40 cycles of 95 °C for 10 s, 58 °C (primers for AOB and total bacteria) or 62 °C (primers for NOB) for 15S, 72 °C for 25 s. The amplification specificities of AOB, NOB, and total bacteria were confirmed by generating melting curves. The standard plasmid DNA were diluted to 1.517×105– 1.517×1010 (total bacteria 16S rRNA), 1.946×105–1.946× 1010 (AOB 16S rRNA), 1.41×105–1.41×1010 copiesμL−1 (NOB 16S rRNA), respectively. The PCR efficiencies were 98.5 % for total bacteria, 89.2 % for AOB, and 105.6 % for NOB, respectively.
All measurements mentioned above were replicated for three times. The data utilized for the analysis were calculated from the median of three paralleled measurements at each harvest time. The treatments of ROX and quantities data of AOB and NOB were analyzed by correlation analysis.
Results Quantities of AOB, NOB, and total bacteria in pot-culture experiments The relationship of environmental variables and ecosystem functioning could be analyzed by the abundances of microorganisms. Under the ROX pressure, the abundances of AOB, NOB, and total bacteria have changed. Meanwhile, earthworms played an important synergistic effect of microorganisms and enhanced microbial diversities. The total bacteria abundances in the group without earthworms were ranged from (2.71±0.45)×108 to (1.49±0.31)×109 copies g−1 dry soil weight, with values 7–22 % lower than those in the group with earthworms [from (2.89±0.28)×108 to (1.92± 0.66)× 109 copies g−1 dry soil weight]. The addition of earthworms increased the abundance of AOB from 8.8 to 37 %, which ranged from (5.84±0.62)×105–(5.20±0.62)× 106 to (6.40±1.06)×105–(8.29±0.86)×106 copies g−1 dry soil weight, while the abundance of NOB increased from (4.13± 0.54)×105–(6.90±1.98)×106 to (4.96±0.83)×105–(8.91± 0.95)×106 copies g−1 dry soil weight, the increase rate was from 16.73 to 22.56 % (Fig. 1). Meanwhile, comparing with the control, the quantities of AOB, NOB, and total bacteria in other treatments were changed evidently (Fig. 1). In two groups without and with earthworms, the quantities of total bacteria in light, medium, and heavy polluted treatments were gradually increased compared to the control, although the quantities of AOB and NOB changed differently. The addition of ROX in light polluted treatments (0.20 and 0.50 mg kg−1) decreased the quantities of AOB by 73–78 % in treatments without
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Appl Microbiol Biotechnol (2014) 98:263–272
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Fig. 1 Abundances and quantities of total bacteria, AOB, and NOB in different treatments. 16S means the total bacteria. 16S, AOB, and NOB present the group without earthworms. 16S+, AOB+, and NOB+ present
the group with earthworms. a–h and i present time profiles of 7, 21, 35, 44, 65, 77, 91, 104 and 140 days
earthworms and 80–85 % with earthworms, while the decrease in NOB quantities was 45–52 % without earthworms and 62–69 % with earthworms. In medium- and heavy-polluted treatments (1.00, 2.00, and 10.00 mg kg−1), the addition of ROX decreased the quantities of AOB by 57, 61, and 66 % in treatments without earthworms and 64, 71, and 76 % with earthworms, in contrast to AOB, the quantities of NOB were increased by 18, 29, and 12 % in treatments without earthworms and 14, 15, and 46 % with earthworms, respectively. Moreover, AOB in the control treatment was significantly higher (p =0.000) than that in other treatments without and with earthworms (Table 1), while NOB in medium and heavy treatments (1.00, 2.00, and
10.00 mg kg−1) were significantly correlated to other treatments (p =0.000; Table 2). Ratios of AOB and NOB in different treatments It is not well established whether there is a ratio of AOB to NOB responsible to the good performance of nitrification, yet the ratio of AOB/NOB is 2.00 through theoretical methods (Hagopian and Riley 1998; Hooper et al. 1997). The reason is that the energy generated from ammonia oxidization by AOB is higher than that produced from nitrite oxidization by NOB. Then, some other studies indicated that a ratio of AOB/NOB of 2.00–3.50 was appropriate related to the inherent high
Appl Microbiol Biotechnol (2014) 98:263–272 Table 1 Statistical evaluation of the quantities of AOB and AOB+ in different treatments by the correlation analysis
AOB presents the group without earthworms. AOB+ presents the group with earthworms.
Group
267 Concentration (mg kg−1)
0 AOB
0
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AOB+
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0.000 0.000 0.000 0.000 0.000 1.000 0.000 0.000 0.000 0.000 0.000
a
The p values show the impact of the ROX treatments on the quantities of AOB and AOB+. Italicized values indicate significant effects (p
