Sludge Bio-Drying Followed by Land Application Could Control the Spread

Environment International 130 (2019) 104906

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Sludge bio-drying followed by land application could control the spread of T antibiotic resistance genes ⁎ Junya Zhanga,b,c, , Qianwen Suia,b,c, Tiedong Lud, Hui Zhonga,b,c, Peihong Shend, ⁎ Yuansong Weia,b,c, a State Key Joint Laboratory of Environmental Simulation and Pollution Control, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China b Department of Water Pollution Control Technology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China c University of Chinese Academy of Sciences, 100049 Beijing, China d College of Life Science and Technology, Guangxi University, Nanning 530005, Guangxi, China

ARTICLE INFO ABSTRACT

Handling Editor: Yong-Guan Zhu The process of sludge bio-drying has been adopted in response to the increasing amount of residual sewage Keywords: sludge. It has been demonstrated that sludge bio-drying effectively reduces both antibiotic resistance genes Bio-drying (ARGs) and mobile genetic elements (MGEs), whereas ermF, tetX, and sulII become enriched in response to the Sewage sludge dynamic development of the microbial community. The present study further demonstrated that the land ap- Antibiotic resistance genes plication of sludge bio-drying products under current application rate did not cause an increase in the abundance Land application of quantified ARGs in the soil but the persistence of ARB should be paid attention. Although land application Soil types introduced ermF, tetX, and tetG into the soil, these soon decreased to control levels. Furthermore, the decay rate varied between soil types, with red soil being the most persistent based on kinetics modeling. The fate of ARGs could also be attributed to the dynamics of the microbial community during land application, and the genus Parasegetibacter, which can degrade extracellular DNA, might play a key role in the control of ARGs. In summary, sludge bio-drying following land application could constitute an effective means of controlling the spread of ARGs, and microbial community changes contributed the most to the fate of the ARGs during the entire treatment chain (residual sewage sludge → bio-drying process → land application).

1. Introduction 2015). Sewage sludge, as an ever-increasing by-product of the wastewater Antibiotic resistance is included in the Frontiers 2017: Emerging treatment process, is frequently used as a soil fertilizer and for soil Issue of Environmental Concern of the United Nations Environment remediation purposes, as it can increase soil organic matter and recycle Programme (UNEP), and antibiotic resistance genes (ARGs) that occur nutrients to the soil (Charlton et al., 2016; Urra et al., 2019). None- in the environment, such as in soils, are currently regarded as a serious theless, it is also an important reservoir of ARGs regardless of the global health concern (UNEP, 2017). Once ARGs are acquired by bac- wastewater treatment process and could introduce a high amount of teria in the environment, the probability of their maintenance in nat- ARGs and antibiotic-resistant bacteria (ARB) into agricultural systems, ural ecosystems is high, even in the absence of high loading of anti- which is particularly concerning due to the global spread of ARGs (Le biotics (Collignon et al., 2018), because there existed many other et al., 2018; Liao et al., 2018; Zhang et al., 2018b; Zhu et al., 2018). It is stressors that could co-select the ARGs, which resulted in an increased thus critically important that sewage sludge is treated properly prior to probability of the ARGs being transmitted and shared between en- land application (Chen et al., 2016). Treated sewage sludge, often re- vironmental bacteria and human pathogens (Burch et al., 2014, 2017; ferred to as biosolids, is subjected to additional processes (such as Luo et al., 2014; J. Zhang et al., 2015; Q.Q. Zhang et al., 2015; Zhou aerobic or anaerobic digestion, composting, and bio-drying) prior to et al., 2018). Soils, as important reservoirs of environmental bacteria, land application in order to minimize the content of pathogenic bac- have been paid increasing attention concerning the spread of ARGs in teria and improve the degree of maturation (Bondarczuk et al., 2016). the environment (Forsberg et al., 2012, 2014; Nesme and Simonet, The treatment of residual wastewater solids could be an excellent

⁎ Corresponding authors at: Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China. E-mail addresses: [email protected] (J. Zhang), [email protected] (Y. Wei). https://doi.org/10.1016/j.envint.2019.104906 Received 6 March 2019; Received in revised form 6 June 2019; Accepted 6 June 2019 Available online 13 June 2019 0160-4120/ © 2019 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/). J. Zhang, et al. Environment International 130 (2019) 104906 opportunity to mitigate the release of ARGs into the soil, and it was use. The basic characteristics of the sludge bio-drying products and soils previously shown that sludge bio-drying could effectively reduce both are listed in Table S1. Soils without sludge bio-drying products were ARGs and mobile genetic elements (MGEs) compared to other tech- regarded as the control group, and the group with the addition of nologies such as anaerobic digestion and composting (Burch et al., chemicals was treated as the NPK group. The urea (47.0 mg/pot),

2017; Zhang et al., 2016b). However, the degree to which sludge bio- NaH2PO4·2H2O (0.73 g/pot), and K2SO4 (0.46 g/pot) were added to drying affects the fate of ARGs in the soil following the land application achieve the final concentration of total nitrogen, 150 mg/kg DW;P2O5, of sludge bio-drying products is unknown. 150 mg/kg DW; and K2O, 100 mg/kg DW. Thus, a total of 27 micro- Inconsistent results exist concerning the fate of ARGs during the cosms were established, and Milli-Q water was used for irrigation land application of treated sewage sludge (Urra et al., 2019; Zhang throughout the experiments. et al., 2018a). For instance, the abundance of ARGs increased following sewage sludge composting, and the land application of sludge composts 2.2. Sample collection and physicochemical parameters further increased the abundance of ARGs in the soils even after 173 days (Zhang et al., 2018a). In contrast, in soil microcosms amended The experiment lasted for 172 days, and samples were collected on with municipal wastewater solids, macrolide resistance gene erm(B) days 1, 6, 12, 22, 43, 82, 117, and 172. Sampling was conducted at a was found to attenuate significantly in around two weeks (Burch et al., depth of 10–15 cm, and three random sites were chosen in each pot. 2014). In one study, wastewater solids treatment technologies, in- The soils were mixed thoroughly, and about 5 g of soil was collected cluding air drying, aerobic digestion, mesophilic anaerobic digestion, from each pot. Soils from the triplicate pots for each treatment were thermophilic anaerobic digestion, pasteurization, and alkaline stabili- then combined as a representative sample. There were 72 samples zation, were used to decrease the persistence of ARGs and intI1 during collected in total, and pH, total organic carbon (TOC), moisture con- their subsequent application to the soil (Burch et al., 2017). Sludge bio- tent, volatile solids (VS), heavy metals, and the C/N ratio were de- drying varied significantly from air drying, and sludge bio-drying isto termined as previously described (Zhang et al., 2016b; J. Zhang et al., treat the sludge with high moisture content (MC) through the aerobic 2015; Q.Q. Zhang et al., 2015). The results are shown in Table S2. degradation of organic substances along with aeration and turning to evaporate water, while air drying is accomplished by loading waste 2.3. DNA extraction and microbial community analysis sludge to an outdoor drying bed. However, other studies have indicated that the long-term field application of sewage sludge increases the Total genomic DNA was extracted in triplicate from 0.5 g of samples abundance of ARGs in the soil (Chen et al., 2016; Xie et al., 2016). The using the FastDNA Spin Kit (MP Biomedical, France) for soil. The tri- inconsistencies between previous studies could be associated with the plicate DNA extracts were then combined to minimize the standard soil types, which might significantly influence the fate of ARGs(Munir deviation due to the operation for further analysis. Extracted genomic and Xagoraraki, 2007; Zhang et al., 2018a), and the treatment process DNA was detected and quantified by electrophoresis on a 1% agarose could also influence the persistence of ARGs during subsequent land gel and a NanoDrop 2000 (Thermo Scientific, USA), respectively, and application. Thus, the role of soil types in the land application of sludge then stored at −20 °C before experimentation. PCR primers 515F/806R bio-drying products in terms of the fate of ARGs should be investigated. targeting the bacterial and archaeal 16S V4 genomic region were se- Ecological succession in the soil resistome is related to a range of lected for microbial community analysis (Caporaso et al., 2010). Small- factors, such as the exogenous loading of ARGs and ARB, varying of the fragment library construction and paired-end sequencing were con- microbial community by organic matter introduction, changes in en- ducted on an Illumina MiSeq sequencing system (Illumina, USA) at the vironmental variables, co-selection from heavy metals, and horizontal Beijing Genomics Institute (BGI; Beijing, China). Pairs of reads from the gene transfer (HGT) (Chen et al., 2017; Tang et al., 2015; Xie et al., original DNA fragments were merged using FLASH (Magoč and 2016, 2018; Zhao et al., 2017). Understanding the influences of these Salzberg, 2011) and then filtered using QIIME quality filters. PCR chi- factors in the fate of ARGs would be of great use in the control of the meras were filtered out in UCHIME (Edgar et al., 2011), following spread of ARGs. Thus, a soil microcosm was established in order to which the sequences were uploaded to MG-RAST (http:// simulate the land application of sludge bio-drying products in three soil metagenomics.anl.gov). The project numbers were 17,760, 17,764, types (red soil, loess, and black soil), clarify the fate of ARGs and ARB, and 17,771 for the NPK, control, and SBD samples, respectively. evaluate the roles of the different soil types, and determine the major Taxonomic classification of the sequences in each sample was per- factors influencing the fate of ARGs. Kinetic modeling was also usedto formed individually using the Ribosomal Database Project (RDP) compare the variance of the ARG decay in the different soil types. Classifier, and the sequences of different taxonomic levels wereas- signed at a bootstrap cutoff of 50% as suggested by the RDP(Wang 2. Materials and methods et al., 2007). Then, the operational taxonomic units (OTUs) with abundances below 0.01% were removed from further analysis. 2.1. Microcosm establishment 2.4. ARG quantification and ARB cultivation Soil microcosms were constructed in triplicate for each group as described previously (Zhang et al., 2018a). Briefly, triplicate micro- Typical ARGs (tetG, tetM, tetX, ermB, ermF, ereA, and blaTEM), the cosms used for each experiment were composed of a mixture of 2.5 kg integron I gene (intI1), four heavy metal resistance genes (MRGs, arsC: soil and about 9.0 g (dry weight, DW) of sludge bio-drying products resistance to As; merA: resistance to Hg; czcA: resistance to Co/Zn/Cd; (SBD) to achieve a total nitrogen content of 150 mg/kg DW for two and pcoA: resistance to Cu), and 16S rRNA were quantified on an ABI rounds of radish planting, and radish was planted to provide the in- Real-time PCR system 7500 (ABI, USA) in triplicate for each sample tegrated soil agro-habitats. Three distinct soil types were selected, in- using a standard curve and a negative control. Detailed information of cluding red soil (S1) from Jiangxi Province, loess (S2) from Shanxi the reaction systems and conditions was provided previously (Zhang Province, and black soil (S3) from Jilin Province in China. The soil et al., 2016b), and the primers and annealing temperature for the textures differ remarkably from each other, as described previously quantitative (q)PCR are summarized in Table S3 with the corresponding (Zhang et al., 2018a). Sludge bio-drying products were collected from a amplification efficiencies and detection limits shown in Table S4.Total sewage sludge bio-drying plant in Shenyang, China, and the technical heterotrophic bacteria (TH) and bacteria resistant to ampicillin (AMP), process was described previously (Zhang et al., 2016b; J. Zhang et al., cefotaxime (CEP), erythromycin (ERY), and tetracycline (TET) were 2015; Q.Q. Zhang et al., 2015). Following air-drying, the soils and determined through cultivation in R2A medium as suggested previously sludge bio-drying products were sieved through a 2-mm mesh before (Zhang et al., 2018a).

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2.5. Data analysis shown in Table 1, and the different persistence of genes base on relative abundance and gene copies is associated with the half-life of the bio- The antibiotic resistance rate was defined as the number of cultur- mass reflected by the 16s rRNA in the soils. The sludge bio-drying able bacteria under the selection of one antibiotic divided by the products did not introduce intI1 and MRGs into the soils, which in- number of total heterotrophic bacteria. The gene copies indicated the dicated the effective reduction of MGEs and co-selection of heavy me- absolute copy numbers present per unit of dry weight (DW), while the tals through sludge bio-drying. normalized copy number by 16S rRNA was regarded as the abundance. The Spearman correlation was performed, and a p value < 0.05 was 3.2. Fate of ARB during the land application of sludge bio-drying products considered statistically significant. The significance of the variance of the dynamics of ARGs between treatments is conducted through the High amounts of ARB existed in the sludge bio-drying products and One-way ANOSIM based on Bray-Curtis. Principal component analysis were generally 3 logs higher than the soils (Fig. S2). The amounts of (PCA), Procrustes analysis, redundancy analysis (RDA), and partial RDA AMP, ERY, and TET were higher than CEP in the sludge bio-drying were conducted using Canoco 5.0 (Microcomputer Power, USA). The products, while TET and ERY were generally lower in the soils (Fig. S2). Mantel test was carried out using PAleontological STatistics software The land application of sludge bio-drying products also introduced (PAST 3.07), and heatmaps were constructed using Heml 1.0 (Deng amounts of ARB (Fig. 2). Compared to the control and NPK, sludge bio- et al., 2014). Network analysis based on the Spearman analysis drying predominantly introduced ERY and TET, which was in ac- (p < 0.01) between ARGs and the microbial community (based on cordance with the entry of ermF, tetG, and tetX. ERY and TET were OTUs), MRGs, and mobile genetic elements (MGEs) was conducted 32.06–34.48 and 35.43–159.50 times higher than the control in the using Gephi (Bastian et al., 2009). ARG data from the soil microcosms soils of D1. The introduced ARB also decreased substantially with land were fitted to a modified Collins–Selleck model (1) and a first-order application, but unlike the fate of the ARGs, which decreased to the model (ExpDec1) to compare the ARG decay between soil types control level, most of the ARB persisted in the soils and differed be- (Sandberg and LaPara, 2016; Zhang et al., 2016b). tween the soil types. For instance, AMP, CEP, and ERY were still higher than the control after day 172 in the red soil and loess. In contrast, N log10 = CS[log10 t log 10 b] AMP, CEP, and TET were higher than the control after day 172 in the N0 (1) black soil. Based on this, the land application of sludge bio-drying products did pose certain risk through the persistence of ARB, whereas N is the number of ARG copies at time t, N is the initial quantity of 0 there was no persistent risk of the fate of quantified ARGs. ARGs, ΛCS is the specific lethality coefficient and b is a lag coefficient. 3.3. Dynamics of the microbial community following the land application 3. Results The microbial community in the sludge bio-drying products differed 3.1. Fate of ARGs during the land application of sludge bio-drying products from the soils, and microbial community also varied between the soil types. The dominant phylum was Bacteroidetes followed by The biomass in the sludge bio-drying products (above 1010 gene Actinobacteria and Proteobacteria in the sludge bio-drying products, copies g−1 DW) was much higher than that in the soils (generally about and the abundance of Acidobacteria was very low (0.2%). Nonetheless, 108 gene copies g−1 DW), and the relative abundance of total ARGs was there existed amounts of Acidobacteria (12.9–24.4%) and lower also about 5 logs higher in the sludge bio-drying products, similar to abundance of Bacteroidetes in the soils (Fig. S3). The land application each target ARG (except ereA and ermB) (Fig. S1). The relative abun- of sludge bio-drying products significantly influenced the dynamics of dance of ARGs in the sludge bio-drying products was 0.48, and it was the microbial community (Fig. 3). generally about 0.003–0.007 in the soils (Fig. S1). The land application The dominant genera in the sludge bio-drying products included of sludge bio-drying products introduced amounts of ARGs regardless of unclassified_Bacteroidetes, Parasegetibacter, Sphaerobacter, Mycobacte- the relative abundance or gene copies (Fig. 1), but these were quickly rium, Castellaniella, unclassified_Flavobacteriaceae, unclassified_ reduced to the NPK and control levels. Furthermore, soil types influ- Chitinophagaceae, Streptomyces, unclassified_Actinobacteria, and enced the decay of the ARGs. ARG abundance was lower than the Aciditerrimonas. The relative abundance of these genera was generally control and NPK levels after day 12 in the loess and black soil, and after above 2.8% in the sludge bio-drying products, but below 1.0% in the day 82 in the red soil, which indicated that sludge bio-drying had a soils. For instance, the abundance of Parasegetibacter was 7.2% in the greater influence on red soil with regards to the fate of ARGs. Thefirst- sludge bio-drying products, but 0.03% in the soils, and the abundance order modeling indicated that t1/2 in the red soil was 46.77 days, but in the soils was higher than 2.7% following land application. Most of 1.63 and 2.15 days for the loess and black soil. The modified Col- these genera could not adapt well to the soil environment and de- lins–Selleck model also confirmed this (Table 1), where the b-values creased to control levels at day 6, except for unclassified_ were 0.78, 0.19, and 0.43 days for red soil, loess, and black soil, re- Chitinophagaceae, which persisted in the soils. The dominant genera in spectively. the soils typically recovered after day 6. The dominant genera were As indicated in our previous study (Zhang et al., 2016b), although Gp4, Massilia, Nitrososphaera, and Nocardioides in the red soil, and the sludge bio-drying could effectively reduce most ARGs and MGEs, ermF abundance of these genera recovered after day 6, as did the dominant and tetX were largely enriched, and the relative abundance of tetG was genera in the loess and black soil (Fig. 3). There were also some genera also much higher in the sludge bio-drying products. Thus, the land present that were not dominant in the soils or sludge bio-drying pro- application of sludge bio-drying products mainly introduced ermF, tetX, ducts but became dominant during application. For instance, Massilia and tetG into the soils. The relative abundance of ermF, tetX, and tetG in became dominant in the loess between days 6 and 43 but was reduced D1 was 28.0–63.5, 5.7–58.6, and 64.9–714.7 times higher, respectively, to the control level at the end, and Janthinobacterium and Pedobacter compared to the control soils, and the enrichment index was much showed a similar pattern. The abundance of Ohtaekwangia in the sludge higher concerning the gene copies of these target ARGs (Fig. 2). Con- bio-drying products and loess was very low, but it became dominant versely, the other target ARGs did not increase along with the land after day 82. Although the land application of sludge bio-drying pro- application of sludge bio-drying products. Generally, ermF, tetX, and ducts disturbed the dynamics of the microbial community significantly, tetG decreased rapidly to NPK and control levels, and the decay rate of they were generally restored to the control and NPK levels, as shown in these ARGs differed between the soil types (Table 1). The ermF persisted Fig. 3. in the red soil, whereas tetX was more persistent in the black soil, as Lefse analysis identified the genera that could act as biomarkers,

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Fig. 1. Dynamic changes in the total ARGs in the different treatments. Seven representative ARGs are quantified in this study; Soils with or without sludge bio-drying products were regarded as SBD and Control, and the group with the addition of chemicals was treated as NPK. thus reflecting the dynamics of the microbial community under theland and black soil, respectively. There were also other biomarkers common application of sludge bio-drying products (Fig. 4). The possible bio- to the soils. The genera Parasegetibacter, Mycobacterium, Sphaerobacter, markers varied significantly between the different soil types. Nineteen and Castellaniella acted as biomarkers in the three soils and were also genera existed that could act as biomarkers for the SBD group in the red the dominant genera in the sludge bio-drying products. These four soil, whereas there were 12 and 11 genera in the loess and black soil, genera should be focused on in the application of sludge bio-drying respectively, that could act as biomarkers for the SBD group. This in- products. Notably, the biomarkers all originated from the sludge bio- dicated that the land application of sludge bio-drying had a greater drying products and not the soils, and the abundance of these bio- influence on the red soil. PCA analysis also indicated that the dynamics markers in the sludge bio-drying products was much higher than in the of the microbial community in the red soil differed significantly from soils. This indicated that the dynamics of the microbial community the loess and black soil, while the difference between the loess and could be mainly attributed to the persistence and decay of the microbes black soil was negligible. from the sludge bio-drying products and not the evolution of the mi- Ureibacillus constituted a biomarker in the red soil, whereas crobes in the resident soils as a result of nutrients from the sludge bio- Castellaniella and Streptacidiphilus were potential biomarkers in the loess drying products.

Table 1

Kinetic coefficients for dynamics of the highly introduced ARGs fitted to the modified Collins–Selleck model(ΛCS, b) and first-order model (k, t1/2) during the land application of sludge bio-drying products.a

Relative abundance Gene copies

−1 −1 ΛCS b (d) k (d ) t1/2 (d) ΛCS b (d) k (d ) t1/2 (d)

Red soil ermF −0.90 1.48 0.01 81.69 −1.23 2.04 2.83 0.35 tetG −0.60 0.23 2.96 0.34 −0.93 0.68 0.73 1.37 tetX −1.49 0.40 0.87 1.14 −1.81 0.63 0.46 2.13 ARGs −0.68 0.78 0.02 46.77 −1.0 1.42 2.63 0.38 Loess ermF −1.74 0.74 0.51 1.95 −1.67 0.56 0.66 1.51 tetG −0.85 0.25 0.97 1.04 −0.78 0.12 2.29 0.44 tetX −1.01 0.07 2.66 0.38 −0.93 0.03 2.33 0.43 ARGs −0.77 0.19 0.61 1.63 −0.70 0.08 0.84 1.20 Black soil ermF −1.59 0.69 0.55 1.80 −1.61 0.68 0.51 1.97 tetG −0.87 0.50 0.86 1.17 −0.88 0.48 0.72 1.39 tetX −1.62 0.73 0.27 3.72 −1.63 0.71 0.23 4.32 ARGs −0.96 0.43 0.46 2.15 −0.97 0.41 0.41 2.42 k is the kinetic coefficients, and t1/2 indicates the half-lives of the target genes. a ΛCS is the specific lethality coefficient, and b is a lag coefficient.

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Fig. 2. Heatmap showing the dynamic changes in ARB, ARGs, intI1, and MRGs during the land application of sludge bio-drying products and NPK compared to the control. The values for each treatment were first divided by the value corresponding to the control and thenlog2 transformed. The black field indicated “not detected”. Soils with or without sludge bio-drying products were regarded as SBD, and the group with the addition of chemicals was treated as NPK. TH, AMP, CEP, ERY and TET indicated the total heterotrophic bacteria and bacteria resistant to ampicillin, cefotaxime, erythromycin and tetracycline, respectively.

3.4. Evaluating the factors influencing the fate of ARGs and ARB 4. Discussion

It was previously documented that microbial community, HGT, co- The treatment rates of domestic wastewater have increased along selection from heavy metals, and environmental variables could influ- with the production of residual sewage sludge, and the appropriate ence the evolution of antibiotic resistance in the environment (Liu treatment and disposal of this sludge has become a major challenge et al., 2018a,b; Luo et al., 2017; Wang et al., 2018). Network analysis (Yang et al., 2015). Land application as the final destination of sewage was conducted based on the correlation analysis between the dynamics sludge has been paid increasing attention along with ARG reduction of the ARGs and these factors (Fig. 5). The ermF, tetG, and tetX possessed (Bondarczuk et al., 2016). However, regardless of the process adopted, the most potential hosts, and most of the hosts originated from the various amounts of ARGs exist in the sewage sludge, necessitating that sludge bio-drying products and acted as biomarkers in the soils during treatment processes prior to land application be conducted (Chen et al., sludge application, which indicated that the decay of these ARGs could 2016). Composting has generally been used to obtain mature sewage be closely associated with the decline of microbes from the sludge bio- sludge and kill pathogens; however, it was documented that com- drying products. As for HGT, significant correlations existed between posting enriched the ARGs in the sludge, and the land application of intI1 and ereA (p < 0.01), which indicated that ereA might pose an sludge compost also increased the abundance of ARGs in the soils (Su HGT risk. The heavy metals could exert certain selective pressure on the et al., 2015; Zhang et al., 2016a, 2018a). ARGs, but the co-occurrence of ARGs and MRGs was limited, and no Nonetheless, the process of sludge bio-drying has increasingly been significant correlation between the environmental variables and ARGs adopted, and the main differences between sludge bio-drying vs. com- was identified. The influence of these factors on the fate ofARBwas posting include the maximum moisture content reduction (reductions also limited. of typically below 40% vs. maintenance at 50–60%, respectively), no A Mantel test based on the Bray-Curtis index indicated that sig- maturation phase (a maturation phase is essential for composting), and nificant correlations existed between microbial community andARG a shorter treatment period (10–25 days vs. 30–50 days, respectively) (J. fate (p = 0.0001, R = 0.4328), and Procrustes analysis also confirmed Zhang et al., 2015; Q.Q. Zhang et al., 2015). Additionally, it has been the significant correlation. This suggested that microbial community demonstrated that sludge bio-drying could effectively reduce both dynamics contributed to the fate of the ARGs during the application of ARGs and MGEs, whereas ermF, tetX, and sulII become enriched due to the sludge bio-drying products. The RDA indicated that these factors the dynamic development of the microbial community rather than HGT (microbial community, intI1, MRGs, and environmental variables) (Zhang et al., 2016b). This study further demonstrated that the land could explain 78.9% of the variance in the fate of the ARGs, and mi- application of sludge bio-drying products did not result in an increase in crobial community dominated these factors based on the partial RDA the abundance of ARGs in the soils. Although land application in- (pRDA), which confirmed the dominant contribution of the microbial troduced certain amounts of ermF, tetX, and tetG into the soils, they soon community to the fate of the ARGs. The RDA also showed the different decreased to the control level, and the decay rate varied between the contributions of these factors to the specific ARGs. Firmicutes, soil types, with red soil being the most persistent. The fate of the ARGs Bacteroidetes, and Chloroflexi had a greater contribution to the fateof could also be attributed to the dynamics of the microbial community ermF, tetX, and tetG, which were highly introduced into the soils during land application. Thus, it was assumed that sludge bio-drying through the sludge bio-drying products. following land application could constitute an effective means of controlling the spread of ARGs, and the microbial community changes

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Fig. 3. Evolution of the microbial community during the land application of sludge bio-drying products in different soils at the genus level as depicted in the heatmap (left) and PCA (right) analysis. For thePCAanalysis, the left and right triangle indicated the samples in the Control and NPK group, the circles indicated the samples from the SBD group, and the color indicated the corresponding soil types. J. Zhang, et al. Environment International 130 (2019) 104906

Fig. 4. Lefse analysis showing the biomarkers during the land application of sludge bio-drying products in different soils. Soils with or without sludge bio-drying products were regarded as SBD and Control, and the group with the addition of chemicals was treated as NPK. contributed the most to the fate of the ARGs during the entire treatment influenced the evolution of the microbial community and thus impacted chain (residual sewage sludge → bio-drying process → land application) the fate of the ARGs indirectly. rather than other factors such as HGT, co-selection from heavy metals, From the perspective of the changes in the microbial community, or environmental variables. Other factors could certainly have the fate of the ARGs is of particular interest. The dominant phyla in the

Fig. 5. Network analysis (A) showing the potential hosts of the ARGs and the significant correlation between the ARGs and influencing factors (p < 0.01). Redundancy analysis (RDA) (B) showing the factors influencing the specific ARGs. EV1 indicated the first principal axis of the environmental variables basedonPCA analysis.

7 J. Zhang, et al. Environment International 130 (2019) 104906 residual sewage sludge were Firmicutes and Proteobacteria, which were indicated that the survival of the ARB introduced into the soil with the also the main hosts of the ARGs, and most of the microbes constituted sludge bio-drying products, not the transient enrichment of indigenous mesophilic bacteria (Ju and Zhang, 2014). A typical bio-drying process resistant strains, contributed to the ARB persistence in this study. would go through a thermophilic period, which inevitably kills or Nonetheless, it should be noted that ARGs, not ARB, were targeted as suppresses most of the mesophilic bacteria and improves the pro- the emerging pollutants in the environment (Pruden et al., 2006). liferation of thermophilic bacteria such as Ureibacillus at the thermo- Furthermore, various environmental microbes constituted potential philic phase (J. Zhang et al., 2015). From the perspective of the char- ARB based on the network analysis, which is particularly concerning, as acteristics of the ARGs, it was hypothesized that ARG proliferation is the ARGs they carried could be transferred into human pathogens and dependent on the microbes, but its reduction is mainly dependent on not the ARB themselves. the environment. The death of the hosts releases ARGs into the en- The main findings of this study are that sludge bio-drying followed vironment, following which these ARGs are then degraded or persist in by land application could constitute an effective means of controlling the environment (Mao et al., 2014). The dominant thermophilic bac- the ARGs originating from sewage sludge, and the changes in the mi- teria grew in the absence of these ARGs, and thus these ARGs could be crobial community contributed the most to the fate of the ARGs during reduced. However, the thermophilic bacteria could have carried other the entire treatment chain. However, the ARGs here constitute the total ARGs, such as ermF, tetX, and sulII, which could ultimately enrich these target ARGs in the soil samples and were not divided into extracellular ARGs. At the thermophilic phase, factors such as selective pressure from and intracellular components. The characteristics and fate of these two antibiotics, co-selection from heavy metals, and HGT had very little components of ARGs obviously differed (Liu et al., 2018a,b). The ex- influence in shaping the microbial community, whereas temperature tracellular ARGs could become intracellular ARGs through transfor- was found to be a key variable (Nakasaki et al., 2009). mation, and intracellular ARGs can be released and become extra- Along with the bio-drying process, the aeration was increased, the cellular ARGs when the hosts die (Zhang et al., 2013). The persistence moisture content was reduced, and the temperature decreased to the and proliferation of intracellular ARGs depend on the hosts, while the environmental conditions (Cai et al., 2013). Unlike composting, the persistence and degradation of extracellular ARGs rely on the sur- thermophilic phase is a physical process rather than a biological process rounding micro-environments (Liu et al., 2018a,b). The dominant genus (Winkler et al., 2013), and moisture content ultimately became the Parasegetibacter in the sewage sludge bio-drying products could have dominant factor, while the influence of other factors was limited. Mi- degraded the extracellular ARGs, which might explain the effective crobes that could endure a lower moisture content (< 40%) grew ra- reduction of ARGs by sludge bio-drying. pidly, including Parasegetibacter, Sphaerobacter, and Castellaniella. Additionally, only a small fraction of known ARGs was investigated Parasegetibacter and Castellaniella were generally isolated from the soil in this study, and other technologies, such as shotgun metagenomics environment (low moisture content environment) (Kim et al., 2015; Lee and high-throughput qPCR, could provide more comprehensive profiles et al., 2010), and Sphaerobacter generally existed in the thermo- of the fate of the ARGs. However, the target ARGs could typically reflect philically treated sewage sludge (Demharter et al., 1989). Interestingly, the change profiles of the fate of the ARGs in the sludge bio-drying the genus Parasegetibacter has the ability to degrade extracellular DNA products, because the enriched ARGs (ermF, tetX), the persistent ARGs (Kim et al., 2015), which could result in a reduction in extracellular (tetG), and the effectively reduced ARGs (blaTEM, ermB) during the ARGs in the environment. This further confirmed the hypothesis that sludge bio-drying products were taken into consideration. the proliferation and death of microbes influenced the fate of the ARGs, and the ARGs released from the death of the microbes were degraded Declaration of Competing Interest effectively during sludge bio-drying. Again, when these microbes en- tered into the soils, particularly those microbes in the sludge bio-drying The authors declare no conflict of interest. products, they could not adapt well to the soil environment. As shown in this study, these microbes decayed, and thus the ARGs they carried Acknowledgement decreased to control levels along with the restoration of the microbial community. It is speculated that the entry of the genus Parasegetibacter This work was supported by the National Major Science & could also assist in the reduction of ARGs due to its ability to degrade Technology Projects for Water Pollution Control and Management of extracellular DNA in the soils, and thus, the ARGs introduced by sludge China (2017ZX07102-002), Natural Science Foundation of Beijing bio-drying products could be effectively reduced. Nonetheless, this Municipality (8182053) and National Natural Science Foundation of needs further investigation to elucidate the speculation. China (51808540). The ARB data collected through the culture-based method (R2A) were comparable with previous studies, and it was previously reported Appendix A. 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