Effects of genetically modified cotton stalks on antibiotic resistance genes, intI1, and intI2 during pig manure composting

Ecotoxicology and Environmental Safety 147 (2018) 637–642

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Effects of genetically modified cotton stalks on antibiotic resistance genes, intI1, and intI2 during pig manure composting

MARK

⁎

Manli Duan, Jie Gu , Xiaojuan Wang, Yang Li, Sheqi Zhang, Yanan Yin, Ranran Zhang College of Natural Resources and Environment, Northwest A & F University, Yangling, Shaanxi 712100, China

A R T I C L E I N F O

A B S T R A C T

Keywords: Antibiotic resistance gene Composting Genetically modified cotton stalk Redundancy analysis

Genetically modified (GM) cotton production generates a large yield of stalks and their disposal is difficult. In order to study the feasibility of using GM cotton stalks for composting and the changes that occur in antibiotic resistance genes (ARGs) during composting, we supplemented pig manure with GM or non-GM cotton stalks during composting and we compared their effects on the absolute abundances (AA) of intI1, intI2, and ARGs under the two treatments. The compost was mature after processing based on the germination index and C/N ratio. After composting, the AAs of ARGs, intI1, and intI2 were reduced by 41.7% and 45.0% in the non-GM and GM treatments, respectively. The ARG profiles were affected significantly by temperature and ammonia nitrogen. In addition, excluding tetC, GM cotton stalks had no significant effects on ARGs, intI1, and intI2 compared with the non-GM treatment (p < 0.05). Thus, similar to non-GM cotton stalks, GM cotton stalks can be used for aerobic composting with livestock manure, and the AAs of ARGs can be reduced. Furthermore, the results of this study provide a theoretical basis for the harmless utilization of GM cotton stalks.

1. Introduction Genetic modification is widely employed to improve production, insect resistance, and disease resistance in cotton (Sun, 2013). The cotton output of China accounts for 25% of the total global cotton production and China is the world's largest cotton producer (Wang et al., 2009), 80% of which is genetically modified (GM) cotton, and the yield of GM cotton stalks is also very large. Cotton stalks contain large amounts of lignocellulose, crude fiber, nitrogen, and phosphorus, thereby making them a promising candidate for use as a feedstock in various processes, especially fertilizer production (Li et al., 2007; Tsai, 2009). Many studies have considered the utilization of cotton stalks. In particular, Mehrdad et al. (2012) investigated bioenergy recovery from cotton stalks by anaerobic digestion process. Moreover, Yuan et al. (2016) studied the anaerobic digestion of cotton stalks by pretreatment with a microbial consortium. However, cotton stalks are generally utilized little and they are mostly burned. Aerobic composting is a promising bioremediation technique (Hamid et al., 2015; Adrian et al., 2016), and it is considered a good method for recycling surplus manure and other materials as a stabilized end-product of agriculture (Bernal et al., 2009). However, there has been little research into the use of GM cotton stalks as an aerobic composting substrate. Therefore, it is

important to examine the feasibility of using GM cotton stalks for composting. Due to the widespread use of antibiotics in animal farms, the environmental contamination caused by antibiotics and antibiotic resistance genes (ARGs) has attracted increasing attention. In addition, the co-selection for heavy metal resistance and ARGs has been investigated widely (Ji et al., 2012; Yin et al., 2017). A large number of ARGs are present in livestock manure, which promotes the horizontal transfer of ARGs in the environment (Maria et al., 2016；Binh et al., 2008). Therefore, the abundance of ARGs should be reduced to prevent their accumulation in livestock manure destined for agricultural use, thereby reducing the environmental risk (Martinez, 2008). Recent studies have shown that composting can be employed to decrease the abundance of ARGs in animal manure. Thus, Chen et al. (2007) demonstrated that composting reduced the abundance of ARGs by several orders of magnitude at the mature stage (i.e., erythromycin resistance genes). Selvam et al. (2012) also indicated that a large number of quinolone, sulfonamide, and tetracycline resistance genes were removed after composting. These studies focused mainly on compost made from livestock manure mixed with wheat or corn stalks. However, the effects of composting with cotton stalks on different types of ARGs are still unclear.

Abbreviations: GM, genetically modified; ARG, antibiotic resistance gene; intI1, Class1 integron gene; intI2, Class2 integron gene; qPCR, quantitative PCR; AA, absolute abundance; RDA, redundancy analysis ⁎ Corresponding author. E-mail address: [email protected] (J. Gu). http://dx.doi.org/10.1016/j.ecoenv.2017.09.023 Received 11 March 2017; Received in revised form 1 September 2017; Accepted 9 September 2017 0147-6513/ © 2017 Published by Elsevier Inc.

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2.3. Sampling

In particular, Zhang et al. (2016a) reported that integron genes facilitate the horizontal transfer of ARGs in microbes and they are considered to be carriers of ARGs. Indeed, many studies have shown that the presence of class1 (intI1) and class 2 (intI2) integron genes may be related to the abundance of ARGs in manure (Zhang et al., 2016b; Chen et al., 2015; Zhu et al., 2013). Consequently, the abundances of integron genes should also be considered when examining ARGs. In this study, we mixed GM cotton stalks with pig manure at a suitable C/N ratio before composting. Our aims were as follows: (1) to investigate the maturation of compost based on changes in its physical and chemical properties; (2) to detect intI1, intI2, and three different types of ARGs in order to estimate the effectiveness of composting at removing ARGs; and (3) to determine the relationships between environmental variables, ARGs, intI1, and intI2. Our results demonstrated the suitability of GM cotton stalks for composting.

Samples weighing ca 1 kg were taken from each treatment on days 0, 2, 4, 7, 14, 26, and 40, where the samples were collected from the top, central, and lower portions of the pile before mixing. The mixed samples were divided into two parts, where one was stored at 4 °C for subsequent physicochemical analysis and the other was freeze-dried using a vacuum freeze dryer (Songyuan, China), before storing at –80 °C prior to use in molecular experiments. 2.4. Analytical methods 2.4.1. Physicochemical analysis and germination test The temperature of the compost pile was measured every day by inserting a mercury thermometer at three holes in each pile. The compost temperature was determined as the average of the three measurements. The moisture content was determined by drying samples at 105 °C for 24 h. The pH, C/N, and germination index (GI) were determined for each compost samples on days 0, 2, 4, 7, 14, 26, and 40 using the following methods. The pH of each compost sample was determined in a water extract with distilled water at a ratio of 1:9 (w/v) using a pH meter (Sartorius, Göttingen, Germany). The total nitrogen contents were determined by the micro-Kjeldahl method. The dried samples were ground and analyzed before determining the total organic carbon content by dry combustion (Zeng and Yu, 2010). The samples were weighed after the loss on ignition in a muffle furnace at 550 °C for 6 h and multiplied by a factor of 0.58 to obtain the organic matter content. The total organic carbon content divided by the total nitrogen content was the C/N value. NH4+-N and NO3–-N were extracted with 2 M KCl and assayed using a segmented flow analyzer (Skalar, the Netherlands) (Santosa et al., 2016). The GI was determined according to Gu et al. (2011). Twenty pakchoi (Brassica chinensis L.) seeds and 5 mL of compost extract were distributed evenly on a sterile culture dish with a filter paper and incubated at 30 °C in the dark for 48 h. Distilled water was used as the control. Each treatment was performed in three replicate dishes. For each treatment, the GI was calculated using the following formula.

2. Materials and methods 2.1. Materials The materials used in this study comprised cotton stalks (non-GM and GM) and pig manure. GM cotton stalks were collected from transgenic Bt (Bacillus thuringiensis) cotton in a plantation in Xinjiang, China. Non-GM cotton stalks were also collected from Xinjiang, China. The cotton stalks were removed from the roots and cut into pieces measuring 2–4 cm in length before composting. The pig manure was collected from a medium-sized dairy farm in Yangling, Shaanxi, China. The fresh pig manure was mixed, air dried to a water content < 30%, crushed, and sieved through a 5-mm mesh. The characteristics of the pig manure and cotton stalks are shown in Table 1.

2.2. Composting The aerobic composting experiment was performed at Northwest A & F University, China, between May 21 and June 29, 2015. The experiment comprised two treatments, where each treatment had three replicates. Composting was performed in six covered bubble boxes, which included a composting chamber (length × width × height = 42 × 42 × 55.5 cm, with a thickness of 3.5 cm). There were three holes (1.5 × 1.5 cm) on the top, bottom, and walls to provide aeration and facilitate the reaction. According to the total carbon and total nitrogen contents of the raw materials, the cotton stalks and pig manure were mixed at a specific proportion to adjust the C/N ratio to 25:1. After mixing, the moisture content of the composting piles was adjusted to 55% by adding tap water. The composting piles were turned over on days 2, 4, 7, 14, 26, and 40 to enhance aeration and to allow sampling. According to the moisture content, more water was added to the compost piles to maintain the moisture at about 55% during the composting process. The composting process was allowed to continue for 40 days.

GI =

2.4.2. DNA extraction and qPCR All of the samples were dried in a freeze dryer (Songyuan, Beijing, China) to ensure that the water content was at the same low level before extracting the DNA. The freeze-dried samples were then crushed and sieved through 1-mm pore filters using an ultra-centrifugal mill (ZM200, Retsch, Germany). Using a FastDNA Kit for Soil (MP Biomedical, USA), DNA was extracted from 0.1 g of each freeze-dried sample according to the manufacturer's instructions. The extracted DNA was stored in a freezer at –20 °C until use. After qualitative detection using standard PCR, nine genes with bright target bands belonging to the three types of ARGs were quantitatively analyzed. The copy numbers of five macrolide resistance genes (erm genes: ermB, ermF, ermQ, ermT, and ermX), two tetracycline resistance genes (tet genes: tetC and tetX), two sulfonamide resistance genes (sul genes: sul1and sul2), and two integrase genes (intI1 and intI2) were determined by qPCR using a BioRad iQ5 Real-Time PCR Detection System (BioRad). Moreover, the 16S rDNA can provide sufficient phylogenetic information regarding the abundance of bacteria in samples (Wang and Qian, 2009). The qPCR reaction system comprised a volume of 20 µL containing 1 µL of DNA template, 10 µL of SuperReal PreMix Plus (TianGen, China), 0.25 µL of each 20 pM primer (ShengGong, China), and 8.5 µL of RNase-free water. The qPCR reactions were conducted under the following conditions: an initial denaturation step at 95 °C for 10 min,

Table 1 Physical and chemical properties of the raw materials used for composting (on an oven dried basis).

Pig manure GM cotton stalks Non-GM cotton stalks

Moisture content (%)

Total carbon (g kg–1)

Total nitrogen (g kg–1)

C/N ratio

pH

7.49 11.13

458.65 558.09

24.58 5.11

18.66 109.22

8.54 6.65

5.46

560.19

6.76

82.87

6.87

Seed Germination × Root Length of Treatment(mm) × 100% Seed Germination × Root Length of Control(mm)

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followed by 40 cycles at 95 °C for 10 s, 60 °C for 30 s, and a final extension at 72 °C for 32 s. The data were collected at 72 °C. Each qPCR reaction was repeated in triplicate. The presence of inhibitory compounds in the extracted DNA was tested by qPCR using serially diluted samples. The limit of qPCR detection for ARGs was 104 copies g–1 compost. The absolute abundances (AAs) of the ARGs were expressed as copies/g of dry compost. We used melting curve analysis to detect nonspecific amplification.

rapidly and reached 60–70 °C in the two treatments, thereby indicating that sugars, proteins, and fats were degraded by microbes (Wang et al., 2016). The maximum temperatures were 72.7 °C and 70.9 °C in the non-GM and GM treatments, respectively. In the two treatments, the thermophilic stage (> 40 °C) was reached within 2 days and it was maintained above 50 °C for around 6 days, as also shown by Malinska et al. (2014), thereby rendering the compost harmless. Thus, both treatments satisfied the standard sanitary requirements. pH is an important factor that affects microbial growth in compost. The changes in the pH under the two treatments are shown in Fig. 1B. During the initial stage of composting, the pH increased rapidly, possibly due to the decomposition of organic acids and the release of ammonia (Liu et al., 2011). The decrease in the pH in the two treatments from 2 to 14 days was probably attributable to the production of organic acids and nitrification (Awasthi et al., 2014). At the maturation stage, the pH values in the non-GM and GM treatments were 8.87 and 8.28, respectively, where a pH of 8.0–9.0 meets the requirements for compost (García et al., 1992). The C/N ratio is an important parameter for assessing the maturity of compost, where it is generally affected by variations in the organic matter content and its characteristics (Mukesh and Akhilesh, 2014). As shown in Fig. 1C, after the high temperature period, the C/N ratio decreased gradually in the two treatments. The final compost product was dark in color with C/N ratios of 19.21 and 17.37 for the non-GM and GM treatments, respectively. Mukesh and Akhilesh (2014) noted that a C/N ratio ≤ 25 is the standard for mature compost. Thus, the two treatments satisfied the maturity requirements and the compost was of good quality (Sharma et al., 1997). These results suggest that GM cotton stalks did not have negative effects on the decrease in the C/N ratio.

2.5. Statistical analysis The standard curves and abundances of ARGs were determined using Microsoft Excel 2013. Changes in the AAs of the ARGs were compared by using SigmaPlot 12.5. SPSS 18.0 was used to test for significant differences between the AAs of ARGs in compost, where differences were considered significant when p < 0.05. Correlations were tested using Pearson's correlation coefficient. Redundancy analysis (RDA) was conducted using Canoco 4.5 (Microcomputer Power, USA). 3. Results and discussion 3.1. Composting process Temperature reflects the microbial activity and progress of composting, and thus it is an important parameter for assessing the maturation of compost (Wang et al., 2011; Tambone et al., 2015). The changes in temperature in the different piles during the composting process are shown in Fig. 1A. Initially, the temperature increased

Fig. 1. Changes in the physicochemical parameters during the critical stages of composting: (A) pile temperature, (B) pH, (C) C/N ratio, and (D) germination index (GI). GM and non-GM denote the addition of GM cotton stalks and non-GM cotton stalks to compost, respectively.

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our trials, and many previous studies have shown that aerobic composting is effective for removing ARGs (Selvam et al., 2012; López et al., 2015; Maria et al., 2016). However, tetC increased in the maturation stage with the non-GM treatment, which does not agree with the results obtained by Zhang et al. (2016c), probably because of differences in the raw material employed and the types of antibiotics added to the feed given to pigs. In the initial sample, sul genes (sul1 and sul2) had the highest AAs, as also shown by Chen et al. (2016), followed by tet genes (tetC and tetX), intI2, and intI1, but erm genes (ermB, ermF, ermT, ermQ, and ermX) were relatively less abundant. Mu et al. (2015) obtained similar results. The AA of sul2 increased during the thermophilic phase in both treatments. Our results are consistent with the observation of Pruden et al. (2012) that tet genes are less recalcitrant than sul genes. Excluding ermT, the other erm genes and tet genes exhibited the same changes as sul genes. Thus, the non-GM and GM treatments resulted in similar changes in the AAs of the tet and sul genes. These similar trends in the AAs were confirmed by the correlation analysis (Table 2), where positive correlations were detected between erm genes, as well as between sul1 and erm genes, and tetX and sul2, possibly because these ARGs had the same host bacteria, or there were positive correlations between their host bacteria (Wu et al., 2015). There were negative correlations between sul2 and erm genes, thereby indicating that there may have been negative correlations between their host bacteria (Qian et al., 2016a). The AA of intI1 exhibited an increasing trend from 2 to 14 days, but at the end of the composting period, the AAs decreased by 29.9% and 13.6% in the non-GM and GM treatments compared with day 0, respectively. It should be noted that the AA of intI2 exhibited a clear decreasing trend throughout the composting process. At the end of composting, the AAs of intI2 decreased by 99.1% and 97.5% in the nonGM and GM treatments, respectively. Similar results were obtained by Qian et al. (2016b). In addition, the AAs of intI1 and intI2 did not differ significantly between the two treatments at the end of composting. Moreover, positive correlations were detected between intI1 and sul1, and intI2 was also positively correlated with tetX (Table 2), which suggests that intI1 and intI2 play important roles in the spread of these ARGs (Chen et al., 2015; Zhang et al., 2017).

Fig. 2. Changes in the absolute abundances of the ARGs, intI1, and intI2 under the two different treatments during the critical stages of composting. G and N denote the addition of GM cotton stalks and non-GM cotton stalks to compost, respectively.

The seed GI is considered to be the most sensitive and reliable index for evaluating the maturity of compost (Zhang and Sun, 2014). As shown in Fig. 1D, the GI was low with both treatments at the beginning of the composting process, but it increased gradually throughout the composting process. If GI ≥ 80% the compost is defined as nonphytotoxic (Fulvia et al., 2015). The GI in the non-GM and GM treatments reached 99.3% and 105.2% after composting for 40 days, respectively, thereby meeting the decomposition requirements. Thus, GM and non-GM cotton both generated suitable compost in terms of seedling root elongation and seed germination (Wang et al., 2015). 3.2. Changes in ARGs, intI1, and intI2 during composting 3.2.1. AAs of ARGs, intI1, and intI2 The AAs of the total ARGs, intI1, and intI2 changed dramatically during composting with cotton stalks (Fig. 2). The copy numbers of each gene are shown in Fig. S1. The AAs of the ARGs in the two treatments increased during the mesophilic and thermophilic stages, where the lowest AAs of the ARGs occurred during the maturation stage. At the end of the composting process, the overall AAs of ARGs, intI1, and intI2 combined were reduced by 41.7% and 45.0% in the nonGM and GM treatments, respectively, which might be related to the effects of temperature on the microorganisms. Most thermophilic microorganisms reproduce rapidly during the mesophilic and thermophilic stages (Białobrzewski et al., 2015), and these thermophilic microorganisms might be the hosts of some ARGs. However, during the maturation stage, these thermophilic microorganisms do not adapt to lower temperatures and they are eliminated, thereby reducing the AAs of ARGs. Most of the ARGs were decreased by aerobic composting in

3.2.2. Differences in the AAs of ARGs after composting Compared with the initial levels, the AAs of ARGs, intI1, and intI2 combined, and 16S rDNA declined from 6.7% to 97.9% and from 4.0% to 97.4% with the non-GM and GM treatments, respectively, after composting for 40 days (Fig. 3). The GM treatment reduced the AAs by 0.67 logs compared with the non-GM treatment. In particular, the AA of tetC declined by 0.46 logs with the GM treatment, but increased by 0.25 logs with the non-GM treatment, which differ from the results obtained by Zhang et al. (2016b). 16S rDNA can provide phylogenetic

Table 2 Pearson's correlation coefficients between the absolute abundances of selected antibiotic resistance genes and those of intI1, intI2, and 16S rDNA.

ermB ermF ermQ ermT ermX sul1 sul2 tetC tetX intI1 intI2 16S rDNA

ermB

ermF

ermQ

ermT

ermX

sul1

sul2

tetC

tetX

intI1

intI2

16S rDNA

1

0.519 1

0.883** 0.812** 1

0.538 –0.041 0.273 1

0.951** 0.630 0.872** 0.523 1

0.513 0.844** 0.714* 0.170 0.711* 1

–0.735* –0.457 –0.749* –0.080 –0.728* –0.495 1

–0.391 0.263 –0.191 –0.258 –0.122 0.518 0.152 1

–0.443 –0.315 –0.445 0.433 –0.394 –0.163 0.704* 0.164 1

0.237 0.598 0.501 –0.014 0.423 0.857** –0.460 0.563 –0.054 1

–0.153 –0.472 –0.277 0.607 –0.273 –0.385 0.493 –0.324 0.820** –0.273 1

0.921** 0.559 0.817** 0.568 0.922** 0.547 –0.729* –0.220 –0.388 0.209 –0.196 1

* Correlation significant at p < 0.05. ** Correlation significant at p < 0.01.

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Fig. 3. Values represent the mean abundances of ARGs, intI1, and intI2 based on three replicates at the end of composting. Bars denote standard errors. The dotted line represents the number of gene copies at the start of composting. *Significant difference at P < 0.05.

information about the bacteria abundance in samples (Chen et al., 2016). We found that the changes in the biomass indicated by 16S rRNA did not differ greatly with the two types of cotton stalks (Fig. S1). Thus, there was no difference between the two treatments in terms of the bacterial abundance. As shown in Table 2, there were positive correlations between 16S rDNA and ermB, ermQ, and ermX. Many studies have found that the fate of ARGs may be influenced by the bacterial community (Zhang et al., 2016c; Cui et al., 2016; Su et al., 2015). In addition, Zhu et al. (2013) found that the composting conditions and the materials used may influence the bacterial communities, thereby explaining the inconsistency between our results and those obtained in other studies. We found that the AAs of most of the ARGs as well as intI1 and intI2 did not differ significantly between the non-GM and GM treatments, except for tetC. However, the AA of tetC decreased after composting with the GM treatment, thereby indicating that it was removed by composting. Thus, the GM cotton stalks had no significant effects on the ARGs, intI1, and intI2 compared with the non-GM treatment. Most of the ARGs were decreased by aerobic composting, which is an effective method for removing ARGs. In addition, we showed that GM cotton stalks are suitable for use in composting.

Fig. 4. Redundancy analysis of the relationships between ARGs and environmental variables in the compost samples on days 0, 2, 7, 26, and 40. The blue arrows indicate the types of ARGs. The red arrows represent environmental variables. Circle symbols represent the samples with the GM treatment. Star symbols represent the samples with the non-GM treatment (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.).

3.3. Relationships between environmental factors and ARGs RDA was conducted to test the relationships between the distributions of different ARGs and environmental factors (Fig. 4). The results showed that the selected variables accounted for 76.4% of the total variation in the changes in the ARGs quantified in this study. Among the selected variables, temperature and ammonium nitrogen significantly explained the changes in genes, i.e., 19.3% and 18.8%, respectively. A previous study by Diehl and La (2010) indicated that temperature is a critical variable with respect to the inactivation and destruction of ARGs and antibiotic-resistant bacteria. Chen et al. (2014) showed that microorganisms use ammonium nitrogen to synthesize important substances during composting. Moreover, Zhang et al. (2016a) showed that the succession of the bacterial community is the dominant factor related to changes in the abundances of ARGs. RDA analysis also showed that pH explained 16.8% of the total variation in the changes in ARGs. Previous studies (Wang et al., 2014; Tang et al., 2015) indicated that some ARGs are associated with the pH and organic matter content, because pH can impose a selective pressure on microbes. The GM and non-GM treatments were grouped together and distributed with the composting period, where there were no differences between the non-GM and GM treatments in terms of the AAs of the ARGs, as well as intI1 and intI2.

them harmless. At the end of composting, the pH (8.87 and 8.28), C/N ratios (19.21 and 17.37), and GI (99.3% and 105.2%) analyses showed that the non-GM and GM treatments satisfied the maturity requirements, and thus the compost was of good quality. In addition, the AAs of most ARGs, intI1, and intI2 were decreased by 41.7% and 45.0% in the non-GM and GM treatments, respectively, after composting for 40 days. The ARG profiles were influenced significantly by temperature and ammonium nitrogen. In addition, GM cotton stalks did not differ in terms of their effects on most ARGs, intI1, and intI2 compared with the non-GM cotton stalks. Thus, similar to non-GM cotton stalks, GM cotton stalks can be used for aerobic composting with livestock manure, where the AAs of ARGs can be reduced. Furthermore, the results of this study indicate that the environmental risk associated with the compost product is reduced, thereby providing a theoretical basis for the harmless utilization of GM cotton stalks.

Acknowledgments This study was supported by the National Natural Science Foundation (41671474 and 41601531), and the 948 Project of Chinese Ministry of Agriculture (2015-Z37). We also thank the anonymous reviewers of this manuscript for their valuable comments.

4. Conclusion The maximum composting temperatures reached in the non-GM and GM treatments were 72.7 °C and 70.9 °C, respectively, which rendered 641

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