Hyphospheric impacts of earthworms and arbuscular mycorrhizal fungus on soil bacterial community to promote oxytetracycline degradation

Accepted Manuscript Title: Hyphospheric impacts of earthworms and arbuscular mycorrhizal fungus on soil bacterial community to promote oxytetracycline degradation Authors: Jia Cao, Chong Wang, Zhengxia Dou, Mengli Liu, Dingge Ji PII: DOI: Reference:

S0304-3894(17)30546-0 http://dx.doi.org/doi:10.1016/j.jhazmat.2017.07.038 HAZMAT 18730

To appear in:

Journal of Hazardous Materials

Received date: Revised date: Accepted date:

6-5-2017 15-7-2017 17-7-2017

Please cite this article as: Jia Cao, Chong Wang, Zhengxia Dou, Mengli Liu, Dingge Ji, Hyphospheric impacts of earthworms and arbuscular mycorrhizal fungus on soil bacterial community to promote oxytetracycline degradation, Journal of Hazardous Materialshttp://dx.doi.org/10.1016/j.jhazmat.2017.07.038 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Hyphospheric impacts of earthworms and arbuscular mycorrhizal fungus on soil bacterial community to promote oxytetracycline degradation

Jia Caoa, b, c, Chong Wanga, b, c*, Zhengxia Doud, Mengli Liua, b, c, Dingge Jia, b, c

a.

College of Resources and Environmental Sciences, China Agricultural University,

Beijing 100193, China b

c

. Beijing Key Laboratory of Biodiversity and Organic Farming, Beijing 100193, China

. Key Laboratory of Plant-Soil Interactions, MOE, Beijing 100193, China

d.

Centre for Animal Health and Productivity, School of Veterinary Medicine,

University of Pennsylvania, 382 West Street Road, Kennett Square, PA 19348, USA

Corresponding author Chong Wang, Ph.D ([email protected] ) Address: 2 Yuanmingyuan Xilu, Beijing 100193, China Telephone: +86 10 62734710; fax +86 10 62731016

Graphical abstract

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Highlights 

AMF hyphae and earthworms accelerated OTC degradation.



Two degradation products were identified as EOTC and ADOTC.



AMF hyphae and earthworm increased bacterial abundance and altered its community.



AMF hyphae and earthworm stimulated certain bacteria, enhancing OTC degradation.

Abstract A two-compartment microcosm was used to investigate the role of arbuscular mycorrhizal fungus (AMF) hyphae and earthworm in altering soil microbial community and OTC degradation. Treatments comprised OTC-contaminated hyphal compartments with or without AMF hyphae and with or without earthworms. Results indicated both AMF hyphae and earthworms accelerated OTC degradation; two degradation products were identified as 4-epi-oxytetracycline (EOTC) and 2acetyl-2-decarboxamido-oxytetracycline (ADOTC). Q-PCR results indicated that both earthworms and AMF hyphae increased 16s rDNA gene, enhancing OTC degradation consequently.

Illumina

sequencing of the 16S rRNA genes showed that AMF hyphae and earthworm altered bacterial community. Earthworms stimulated the growth of class Anaerolineae, family Flavobacteriaceae, Genus Pseudomonas, reducing OTC residues. AMF hyphae significantly increased the abundance of family Pirellulaceae, genus Glycomyces, and Nonomuraea which had a negative correlation with EOTC, accelerating OTC degradation. When used together, AMF hyphae and earthworms enhanced OTC degradation by stimulating class Anaerolineae and family Flavobacteriaceae.

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Keywords: Hyphal compartment; High Throughput Sequencing; Q-PCR; OTC degradation products

1. Introduction

Agricultural soils are a major environmental reservoir for antibiotic residues, as antibiotics are commonly used in livestock farming and much of it eventually ends up in manure which is subsequently applied to agricultural land [1,2]. A large proportion (30-90%) of the antibiotics may be excreted in manure due to incomplete metabolism [2]. These animal wastes containing antibiotics might disrupt certain microbial activities, induce antibiotic resistance gene and affect plant growth [3,4]. Oxytetracycline (OTC) is a tetracycline antibiotic that has been widely used in livestock, poultry, and aquaculture production and in human medicine without strict controls [5]. OTC was found in animal waste at concentrations of mg kg−1 [6]. Additionally, a variety of OTC metabolites such as 4-epi-OTC (EOTC) and α-apo-OTC were detected in interstitial water, soil and animal urine and feces, and the antibiotic activity of EOTC and α-apo-OTC was approximately 30% and 7–10% of OTC, respectively [7,8]. Another degradation product ADOTC was proposed in biodegradation of OTC by pleurotus ostreatus mycelium which has lower antibacterial potency and higher lipophilicity than OTC [6]. Understanding the OTC degradation products could provide more complete understandings of OTC degradation. To avoid the negative effects, bioremediation techniques are possible solutions for eliminating or reducing the antibiotic from soil. Arbuscular mycorrhizal fungi (AMF) have been associated with bioremediation processes indirectly due to the so-called mycorrhizosphere (the zone influenced by both the root and the mycorrhizal fungus) effect [9,10]. AMF can stimulate soil microbial activity, 3

shift bacterial community, improve soil structure, and contribute to overall bioremediation of pollutants [11,12,13,14]. The AMF hyphal exudates contain energy-rich low-molecular-weight sugars, organic acids and unidentified high-molecular-weight polymeric compounds, which can stimulate or inhibit hyphosphere bacteria [15]. Few studies have explicitly studied how AMF influence the soil bacterial community responsible for OTC degradation. In our previous study, the inoculation of Rhizophagus intraradices significantly increased OTC degradation [16]. However, we did not separate the mycorrhizosphere and hyphosphere (the hyphae-soil interface) effects in the study. Root exudates provide a nutrient-rich environment where microbial activity is stimulated, which leads to more efficient degradation of pollutants [17]. We found no previous studies investigating the effect of AMF on the hyphosphere soil microbial community mediating OTC dissipation. Earthworms function as ecosystem engineers and have a substantial influence on the fate of organic pollutants in soil [18,19]. They directly and indirectly increase soil aeration, improve the transport and distribution of microorganisms through bioturbation, and enhance the contact between microorganisms and pollutants. Earthworms also increase the microbial population and distribution, which aids in the degradation of organic pollutants [19,20]. The positive effect of earthworms on the removal of organic pollutants has been reported in several studies, earthworms can accelerate the removal of atrazine, PCBs, and PAHs in the soil [19,21,22]. Our previous results indicated that earthworms significantly increased the degradation of OTC [16]. The system which we studied was complex, including plant (root), earthworm, and AMF. However, very little information is available concerning how earthworm affect the microbial degradation of OTC without root, and whether earthworm interacts with AMF hyphal in the hyphosphere.

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Earthworms and AMF are important organisms belonging to different guilds in soil and can create distinct niches [23]. Several studies have reported the positive interactions of earthworms and AMF in influencing soil fertility and plant growth [24]. Earthworms may stimulate and or suppress the growth of microorganisms by feeding, secreting mucus, and burrowing [19]. AMF hyphae release of exudates include formate, acetate, and glucose, which can be readily assimilated by bacteria as carbon sources, leading to a stimulation of some bacteria in the hyphosphere [15]. Additionally, some unidentified or trace substances in the exudates may inhibit bacteria growth [25]. These two taxonomically different soil organisms coexisting in the soil (with root) could interactively enhance OTC degradation [16], but little information is available on the interaction of AMF hyphal and earthworms on the degradation of OTC in the hyphosphere. Hyphosphere is always defined as hyphae-soil interface where roots are absent, higher bacterial abundance and altered bacterial community were established [26]. No study has described bacterial communities in OTC polluted hyphosphere soil. Direct interactions between AMF and other microorganisms (eg. phosphate solubilizing bacterium) have been investigated in hyphosphere [27]. However, little is known about the effect of earthworm and AMF on the soil microbial community mediating OTC degradation in the hyphosphere. There is a close correlation between soil bacterial communities and pollutant degradation [14, 20]. The aim of this study was to investigate how the earthworm and AMF alter the bacterial communities and OTC degradation in the hyphosphere soil. We hypothesized that adding AMF hyphae and earthworm results in different native bacterial community and would therefore accelerate OTC degradation in the hyphosphere soil. To accomplish this, we established two-compartment microcosm units to control AMF hyphae access to the OTC contaminated soil. We studied the OTC degradation and analyzed its products in the hyphosphere.

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We also analyzed the changes in the microbial community structure using 454 pyrosequencing with bacterial-specific primers targeting the 16S rRNA gene associated with earthworm and mycorrhizal hyphae. 2. Materials and methods 2.1 Antibiotic and soil The antibiotic used in the study was OTC (Terramycin®, 200 mg ml-1 base as OTC dihydrate). The soil used in this study was collected from the Shangzhuang experimental station, Beijing (116.25E, 40.13N). The plot had received no fertilizer or manure or any known materials containing antibiotics for at least 10 years. The soil has the following basic physicochemical properties: pH (Soil: H2O, 1:2.5) 7.40, total nitrogen concentration 0.10 %, organic matter concentration 1.25 %, cation exchange capacity 10.43 cmolc kg-1, Olsen-P of 13.65 mg kg-1 and an NH4Cl-exchangeable K 143.0 mg kg-1. The soil was air-dried, sieved (2 mm) and then irradiated before use to eliminate indigenous microorganisms (25 kGy of 60 Co γ-radiation for 72 h at the Beijing Radiation Application Research Centre). 2.2 Earthworm, mycorrhiza and maize The epigeic earthworm species Eisenia fetida was used in our study, whose surfaces or gut contents were pretreated to minimize the impact of naturally occurring mycorrhizal propagules [24]. The AMF inoculum was Rhizophagus intraradices (BEG JX04B), propagated on maize and white clover host plants in a growth chamber at 30°C/18°C with a 16 h/8 h light/dark regime and 50–75% relative humidity. Maize seeds were Zheng Dan 958, which were surface sterilized with a 10% (v/v) solution of H2O2 for 10 min, rinsed thoroughly with deionized water, and then placed on autoclaved filter paper soaked with sterile distilled water and incubated at 25 °C for 24 h [24].

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2.3 Experimental design and incubation We constructed two-compartment microcosm units to investigate the earthworm-AMF interaction on OTC degradation in the hyphal compartment. Each microcosm units comprised two jointed compartments (root compartment 10*10*10, hyphal compartment 9*10*10) separated by a doublemash barrier. The mesh was either 30 μm to allow passage of mycelia but not roots, or 0.45 μm, to exclude both mycelia and roots. Root compartment contained plant, with the hyphal compartment controlling mycelia access to the compartment. The root compartment was filled with 600 g sterilized soil and the mycorrhiza inoculum (consisting of spores, mycelium, fine root segments, and soil) was placed approximately 2 cm under the soil surface at 50 g kg-1, and one germinated maize seed (Zea mays L., Zheng Dan 958) was sown in each root compartment. OTC was spiked into hyphal compartment soil (500 g sterilized soil) by adding an OTC solution to make nominal initial concentrations of 100 mg OTC kg_1 of soil, and then the actual initial concentrations were determined to be 99.3 mg OTC kg_1 (dw) of soil. Four treatments were set up representing all the combinations of AMF (with or without) with earthworms (with or without) in the hyphal compartment: (1) CK, control with neither AMF hyphae nor earthworms in the hyphal compartment; (2) E, with earthworms present and without AMF in the hyphal compartment; (3) AM, with AMF hyphae and without earthworms in the hyphal compartment; and (4) AM+E, both earthworms and AMF hyphae were present in the hyphal compartment. Ten earthworms with similar fresh weights (3.05±0.04 g) were added to hyphal compartment in the treatment of E and AM+E. All treatments received basal fertilization of N, P, K, Mg, Mn, Zn and Cu [28]. Each treatment had three replicates, plants in these microcosms were grown at China Agricultural University in Beijing from 6 September to 31 October 2015 at 24°C:

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30°C (night : day). Throughout the incubation, moisture content was monitored weekly and maintained by adding sterile deionized water when necessary. 2.4 Sampling and analysis At destructive harvest, soil samples were collected from the hyphal compartments, and the top 2 cm of the soil from these samples was discarded to eliminate any possible surface effects. 2.4.1 Determination of AMF colonization, the OTC concentration and OTC products

AMF colonization was determined by the method modified by McGonigle et al. (1990) [29]: 1g of root was cut into 1cm pieces, and thirty random pieces were then cleared in 10% KOH, acidified in 1% HCl, and stained in 0.05% Trypan blue in lactoglycerol. The colonization of each root sample was determined at 250×magnification using the line intersect method. The quantification of OTC was carried out by a high-performance liquid chromatography (HPLC, Agilent 1100 Series, Agilent Technologies, USA). The detailed measurement information can be found in our previous research [16]. Agilent 1200 series HPLC coupled to an Agilent 6420 triple quadrupole mass spectrometer with electrospray ionization mode was used for the detection and identification of degradation byproducts (for details, see Supplementary Material). 2.4.2 qPCR Assay and High Throughput Sequencing Total DNA was extracted from 0.5 g soil using a MoBio Powersoil TM soil DNA isolation kit (MoBio Laboratories, USA) according to the manufacturer’s protocol. The abundance of the bacteria was estimated from real-time qPCR assays targeting 16S genes in soil DNA extracts. The qPCR and standard curve determination were act as described by our previous study [16]. The 16S rDNA high-throughput sequencing was performed by Realbio Genomics Institute (Shenzhen, China) using the Illumina MiSeq platform. The 16S V4 region was amplified using the

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primers 515F (5′-GTGCCAGCMGCCGCGG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT3′) [30]. According to the standard protocols, purified amplicons were pooled in equimolar and paired-end sequenced (2 Х 250 bp) on an Illumina MiSeq platform in Shenzhen, China (details are provided in Supplementary Material). The reads were demultiplexed, quality-filtered, and processed using QIIME [30]. Operational Units (OTUs) were clustered with a 97% similarity cutoff using UPARSE and chimeric sequences were identified and removed using UCHIME. The taxonomy of each 16S rRNA gene sequence was analyzed by RDP Classifier against the Silva (SSU115)16S rRNA database using a confidence threshold of 70%. Finally, the complete dataset was sent to the Sequence Read Archive (SRA) database of the National Center for Biotechnology Information (NCBI). 2.5 Data analysis Data were subjected to analysis of variance of all parameters/response variables, which was carried out using SPSS software, version 17.0 (SPSS Institute, Inc., Cary, NC, USA). Fisher's LSD (Least Significant Difference) test was used to test for differences between treatment means with significance level at the 5%. The resulting distance matrix was visualized using PCoA of the UniFrac distances in QIIME. Processing of OTUs was performed mostly in MOTHUR after the number of sequences was standardized between samples by eliminating sequences with random numbers. OTC degradation byproducts were calculated used the ratio of Ct/C0, where Ct is the concentration of substrate at any given point of time and C0 is the initial substrate concentration. 3. Results 3.1 Earthworm survival and AM colonization Almost all earthworms were collected at maize harvest, and their weight did not change during the

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growing time. Maize root mycorrhizal colonization rate in all treatments ranged from 56.41% to 60.18% after 56 days of growth, with E+AM treatment showing the highest AM colonization. 3.2 The degradation of OTC and its products After a 56-day incubation, the OTC concentration in the hyphal compartment were 33.31 mg/kg, 35.06 mg/kg, and 27.82 mg/kg in E, AM, E+AM treatments, respectively, which were significantly lower than those in the control (p<0.05; Table 1). Additionally, earthworms and AMF hyphal showed a significant interaction on the degradation of OTC (p<0.05). Presence of possible degradation products was detected. The HPLC results showed two partially overlapping peaks (Fig. S1), suggesting the structures of two detected substances. One indicated by an OTC precursor ion [M+H]+ m/z 461, possibly 4-epi- oxytetracycline (EOTC, Fig. S2a), which is the OTC reversible epimer that only differ at the C-4 position of the molecule and thus have very similar MS2 scans. The other indicated by a precursor ion [M+H] m/z 460, which could be the degradation product of OTC. This additional precursor ion could be interpreted as 2acetyl-2-decarboxamido-oxytetracycline (ADOTC, Fig. S2c). The AM and E+AM treatments showed a lower EOTC ratio compared to the control (p<0.05; Table 1). The individual addition of earthworms and AMF significantly increased the ratio of ADOTC (p<0.05; Table 1), indicating that earthworm and AMF could increase the transformation rate of OTC to ADOTC. The interactive effect of earthworms and AMF hyphal on the ratio of EOTC and ADOTC were particularly marked (p<0.001).

3.3 Bacterial 16S rRNA gene abundance In the hyphal compartment, bacterial 16S rRNA gene abundance significantly increased in E, AM, and E+AM treatments compared to the control (p<0.05; Table 1). Additionally, there was a

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significant interaction between earthworms and AMF hyphal on the abundance of bacterial 16S rRNA gene (p<0.01). 3.4 General description of 16S rRNA gene sequences A total of 451,021 bacterial sequences and 10,667 OTUs (at the 3% evolutionary distance) were identified in the present study. A Venn diagram demonstrated that the OTUs differed among the four treatments (Fig. 1). The number of OTUs ranged from 2544 (E+AM treatment) to 2728 (AM treatment). 1780 in 10,667 OTUs were shared by all four treatments. To gain better insight into the differences of soil bacterial community among the four different soil samples we applied heatmap analyses of the most abundant 100 OTUs, which highlighted their relative distributions and abundances (Fig. 2). As shown in heatmap, the abundance of dominant 100 OTUs differed among the four treatments. The dominant OTUs in each treatment were also different. The OTU diversity estimated by Shannon’s diversity index suggested that the bacterial diversity increased significantly in E and E+AM treatments in the OTC polluted soil (p<0.05; Table 1), and the highest soil bacterial diversity was observed in the E treatment, followed by the E+AM treatment. A phylogenetic tree of the majority 10 of the bacterial sequences cluster in the phyla in each treatment were detected (Fig. S3). The results indicated that OTUs belong to Streptomyces, Luteimonas,

Lysobacter,

Nitrososphaera,

Steroidobacter,

Pseudomonas,

Glycomyces,

Nonomuraea,

Bacillus,

Devosia,

SMB53,

Lutibacterium,

Candidatus

Flavobacterium,

Plantocomyces were predominant. Of these OTUs, earthworm caused an increase in abundance of Planctomyces (47 OTUs) and Flavobacterium (13 OTUs). AMF and both AMF and earthworm caused an increase in abundance of Nonomuraea (2 OTUs) and a decrease of Bacillus (5 OTUs)

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(Fig. S3). 3.5 Bacterial community structure according to dominant classes, family and genera

The relative abundance of the top 10 taxonomic categories at class level were summarized (Fig. 3a). The Actinobacteria was largely dominant, representing 14.97-21.20% of the population in all treatments. The Gammaproteobacteria was the next dominant taxonomy, followed by Alphaproteobacteria, Cytophagia, and Planctomycetia. E and E+AM treatment significantly increased the abundance of Cytophagia, Alphaproteobacteria and Flavobacteriia (p<0.05; Fig. 3a). Among the 10 selected classes, the Cytophagia (r2=-0.784, p<0.01), Anaerolineae (r2=-0.706, p<0.05) and Flavobacteriia (r2=-0.817, p<0.01) were found to negatively correlate with the concentration of OTC. Additionally, Gammaproteobacteria (r2=-0.583, p<0.05) and Planctomycetia (r2=-0.650, p<0.05) showed significant correlation with the ratio of EOTC (Table 2). At the family level, Streptomycetaceae, Xanthomonadaceae, Cytophagaceae, Pirellulaceae, and Flavobacteriaceae dominated, among which Streptomycetaceae was the most abundant. E and E+AM treatments significantly increased the abundance of Cytophagaceae, Flavobacteriaceae,

and Alteromonadaceae. Additionally, AM and E+AM treatment significantly increased the abundance of Pirellulaceae (p<0.05; Fig. 3b). The relative abundance of family Cytophagaceae (r2=-0.776, p<0.01), Flavobacteriaceae (r2=-0.830, p<0.01), Alteromonadaceae (r2=-0.675, p<0.05) were negatively correlated with residual OTC. While Streptomycetaceae (r2=0.863, p<0.01) and Xanthomonadaceae (r2=0.872, p<0.01) showed positive correlation with the concentration of OTC. The abundance of Pirellulaceae showed negative correlation with the ratio of EOTC (r2=-0.779, p<0.01; Table 2). At the Genus level, Streptomyces, Pseudomonas, Luteimonas, Lysobacter and Steroidobacter

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dominated, Streptomyces and Pseudomonas are the five most abundant genus. E and E+AM treatments significantly increased the abundance of Pseudomonas, Cellvibrio. The abundance of Nonomuraea significantly decreased in AMF treatment, but increased in E and E+AM treatments (Fig. 3c). The abundance of Pseudomonas (r2=-0.632, p<0.05) and Cellvibrio (r2=-0.765, p<0.01) showed negative correlation with residual OTC. The abundance of Nonomuraea (r2=-0.601, p<0.05) and Glycomyces (r2=-709, p<0.01) showed negative correlation with the ratio of EOTC (Table 2). 3.6 Microbial Community Structure Elucidated by PCoA Figure 5 shows the unweighted PCoA based on the absence or presence of phylotypes. Most notably, E and E+AM treatments have much higher PC1 values compared to those in CK and AM treatments. As with PCoA, the earthworm treatment (E, E+AM) had a distinct community structure and clustered together. The PCoA results strongly indicate that earthworm play an important role in shaping the bacterial community, which is responsible for OTC dissipation. 4. Discussion 4.1 The OTC products and its degradation in hyphosphere Several of the degradation products, such as EOTC and ADOTC, were known to maintain some potency of their parent substance, OTC, on both sludge and soil bacteria. These chemicals have lower toxicity and environmental mobility [7]. Chen et al. [31] showed that the degradation of OTC resulted in an initial rapid increase in EOTC concentration, especially in the high OTC treatment groups, which decreased gradually when EOTC began to break down. In the present study, AM and E+AM treatments significantly decreased the ratio of EOTC at day 56 (Table 1), pointing to more efficient and prolonged decrease of OTC concentrations. Previous studies indicate that OTC was first degraded to EOTC and that the subsequent degradation of EOTC accelerated the OTC

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degradation process [32,33,34]. Lower EOTC with lower OTC at day 56 could point to the essential role of EOTC degradation in enhancing the OTC degradation process. Our results thus reaffirm previous reports that EOTC is the main metabolite of OTC degradation and EOTC’s degradation is essential to enhance OTC degradation. AMF may enhance the degradation of OTC by accelerating the degradation of EOTC. ADOTC is known to be a byproduct of OTC production by Streptomycesrimosus, which has lower antibacterial potency on environmental relevant bacteria and higher lipophilicity than OTC [6,8]. In the present study, earthworm and AMF can increase the transformation rate of OTC to ADOTC, which may fulfill the aim of the bioremediation approach. 4.2 Earthworm induced Microbial Community changes and OTC degradation in the hyphosphere It is clear that earthworms can accelerate the removal of organic contaminants, such as herbicides, PCBs, PAHs, and crude oil [19]. Earthworms enhance hydrocarbon degradation by stimulating microbial growth via excretion of readily degradable carbon, and they also effect on the bacterial community via razing, gut passage [35]. The treatments E and E+AM were differentiated from the other two treatments by positive PCoA value in the present study (Fig. 4), which suggested that earthworms could play an important role in shaping the bacterial community in the hyphosphere. E and E+AM treatments significantly increased the soil bacterial 16S rDNA genes abundance and bacterial Shannon diversity index (Table 1), and the soil residual OTC concentration negatively correlated with the soil bacterial 16S rDNA genes (r2=-0.826, p<0.01) and bacterial Shannon index (r2=-0.638, p<0.05) (Fig. 5). These results indicated that earthworm could enhance the OTC degradation by increasing bacterial biomass and diversity. Previous studies showed that earthworms affected bacterial functional communities and

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organic matter metabolism, and strongly stimulated the growth of several bacterial families, such as Pseudomonadaceae, Flavobacteriaceae, Comamonadaceae, and Sphingobacteriacea [36,37] have revealed Pseudomonadaceae, Flavobacteriaceae, and Comamonadaceae played key roles in 2,4dichlorophenol (2,4-DCP) biodegradation in the earthworm treated soil. Flavobacteriaceae was stimulated by earthworm and thus enhanced the pentachlorophenol degradation in soil [36]. In the present study, earthworm activity stimulated Flavobacteriaceae which had a significantly negative correlation with residual OTC (r2=-0.830, p<0.01 Table 2). This indicated that Flavobacteriaceae were involved in OTC degradation as driversin soils. The relative abundance of family Streptomycetaceae and Xanthomonadaceae positively correlated with the concentration of OTC, as these two families were relatively lower in the treatments (E, E+AM) (Fig. 3b), we can speculate that earthworm may have grazed them or secreted inhibitors that resulted in decreased biomass. Additionally, earthworm treatment can stimulate certain bacteria which may accelerate soil pollutant degradation. Pseudomonas are ubiquitously present and most frequently isolated from the contaminated environments. Some of the Pseudomonas strains are well known to degrade or cometabolize PCB and biphenyl [38]. Monard et al. [39] showed that Pseudomonas was one of the most dominant atrazine degraders in earthworm burrow linings. Singh et al. [40] also found that Pseudomonas strain was likely to be involved in biodegradation of chlorpyrifos in soil. In the present study, Pseudomonas significantly increased in the E and E+AM treatments (Fig. 3c), Pseudomonas showed significant negative correlation with residual OTC (r2=-0.632, p<0.05 Table 2). This suggested that Pseudomonas may be stimulated by the presence of earthworm in OTC polluted soil and resulted in a lower OTC residual. 4.3 AMF hyphae induced microbial community changes and OTC degradation

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AMF produce extensive extraradical hyphae in the soil, providing a habitat for microbes [41]. AMF hyphae function as rapid conduits for recent plant photosynthates, which can attract microbes and stimulate their growth [42]. In the present study, AMF hyphae showed no obvious difference in the microbial community compared with control (Fig. 4), which were consistent with results from previous studies that the microbial community structures in soil accessed by AMF hyphae, but not roots, did not discriminate strongly from those in unplanted soil [43]. Herman et al. [44] also reported that AMF hyphae influence microbial populations in the hyphosphere, through stimulatory and suppressive effects on specific components, but impacts on the whole community structure were relatively minor. AMF hyphae significantly increased the abundance of 16s while having no effect on bacterial Shannon index (Table 1), which indicated that AMF hyphae stimulated bacterial populations in the hyphosphere. Most previous studies have found that AMF likely played a critical role in the degradation of organic contaminants in soils [45]. Similar to roots, AMF hyphae release C-rich compounds into the soil which can stimulate microbial growth and function [46]. However, AMF hyphae do not benefit all microbes, they inhibit some as well [13,47]. AMF to enrich for microbial species in the hyphosphere have been reported previously [43]. In the present study, AMF hyphae significantly increased the abundance of class Anaerolineae, family Pirellulaceae, genus Glycomyces, and Nonomuraea (p<0.05, Fig. 3). The strictly anaerobic genera in Anaerolineae class could ferment carbohydrates and amino acids around the mycorrhizal hyphae, and may also utilize low chlorinated biphenyls such as dichlorinated biphenyls [10]. Anaerolineae negatively correlated with the concentration of OTC (r2=-0.706, p<0.05, Table 2), which indicated that mycorrhizal hyphal exudates stimulated the growth of Anaerolineae and accelerated the OTC degradation. The

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concentration of degradation product EOTC negatively correlated with the abundance of Pirellulaceae (r2=-0.661, p<0.05), Glycomyces (r2=-0.709, p<0.01), and Nonomuraea (r2=-0.601, p<0.05) (Table 2). As the subsequent degradation of EOTC would accelerate the OTC degradation process [34], we can assume that AMF hyphae would stimulate these bacteria in the hyphosphere and accelerate OTC degradation. 4.4 Combined effect of earthworm and AMF hyphae on OTC degradation There has been an increasing focus on complex interactions between AMF and earthworms in the bioremediation of pollutants. Plant-AMF-earthworm association can significantly accelerate the degradation of pollutants [16,45,48,49]. However, less information is known about the specific interaction mechanisms among AMF hyphae and earthworm (without plant roots), particularly in soils contaminated with pollutants. In the present study, the co-existence of AMF hyphae and earthworm significantly increased the abundance of bacterial 16s rDNA and bacterial Shannon index (p<0.05, Table 1). Additionally, the combination of earthworm and AMF hyphae stimulated some bacteria, such as class Anaerolineae, family Flavobacteriaceae. The dual AMF hyphae and earthworm inoculated soils had the lowest OTC concentration and the highest abundance of Flavobacteriaceae (Table 1; Fig. 3). Additionally, earthworms and AMFhyphal showed significantly interactions on residual OTC, the ratio of EOTC and ADOTC, and the abundance of bacterial 16s rDNA (p<0.05, Table 1). The negative correlation between residual OTC concentration, the abundance of bacterial 16s rDNA, bacterial Shannon index, Flavobacteriaceae, as well as the negatively correlation between EOTC and Anaerolineae, we propose that the positive interaction between AMF hyphae and earthworms could enhance OTC degradation in hyphosphere.

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5. Conclusions The present study indicates the co-existence of AMF hyphae and earthworms could interactively enhance the degradation of OTC in hyphosphere soil. Both AMF hyphae and earthworms could stimulate certain bacteria which showed correlation with the OTC and EOTC concentrations. Our study provides clear and strong evidence that earthworm and AMF hyphae could accelerate OTC degradation by altering bacterial growth and community compositions in hyphosphere. These findings improve our understanding of soil biology interactions in OTC degradation. Further studies are needed to explore the direct effect of soil biota on antibiotic resistance genes and the specific interaction mechanisms among soil biology in polluted soil.

Acknowledgments This work was funded by the National Natural Science Foundation of China (Project 31570514), The Da Bei Nong's Youth Researchers Program (2413002), Innovative Group Grant of the National Science Foundation of China (31421092) and National key research and development program (2016YFE0101100). We also acknowledge Dr. Zhenjun Sun and Dr. Xiaolin Li at China Agricultural University for laboratory assistance.

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Figure Captions Fig. 1 A Venn diagram displaying the degree of overlap of bacterial OTUs (at the 3% evolutionary distance) among the different treatment. The OTUs differed among the four treatments, and the number of OTUs were 2709 (CK), 2686 (E), 2728 (AM) and 2544 (AM.E). 1780 in 10,667 OTUs were shared by all four treatments. CK is the soil without the addition of earthworms and AM fungi; E is the soil with the addition of earthworms; AM is soil with the addition of AM fungi; AM.E is the soil with both earthworms and the addition of AM fungi. Fig. 2 A heatmap diagram visualizing the dominant 100 OTUs among the four treatments. The abundance of dominant 100 OTUs differed among the four treatments and the dominant OTUs in each treatment were also different. CK, control with neither AMF hyphae nor earthworms in the hyphal compartment; E, with earthworms present and without AMF hyphae in the hyphal compartment; AM+E, both earthworms and AMF hyphae were present in the hyphal compartment. Fig. 3 The effect of different treatments on the abundance of bacterial groups at the class (a), family (b), and genus (c) level, respectively. The middle lines of the box plots represent median values (n = 3), with bars showing value ranges (minimum to maximum). Different letters indicate significant differences among treatments. CK, control with neither AMF hyphae nor earthworms in the hyphal compartment; E, with earthworms present and without AMF hyphae in the hyphal compartment; AM+E, both earthworms and AMF hyphae were present in the hyphal compartment. Fig. 4 PCoA based on the unweighted UniFrac analysis showing the microbial community grouping. E and AM.E treatments showed obvious difference in the microbial community compared with CK. CK, control with neither AMF hyphae nor earthworms in the hyphal compartment; E, with earthworms present and without AMF hyphae in the hyphal compartment; AM.E, both earthworms

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and AMF hyphae were present in the hyphal compartment. Fig. 5 Correlation between residual OTC concentration and the soil bacterial 16S rDNA genes and bacterial Shannon index. Residual OTC concentration had a significant correlation with the soil bacterial 16S rDNA genes (r2=-0.826, p<0.01) and bacterial Shannon index (r2=-0.638, p<0.05).

Fig. 1

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Fig. 2

28

Fig. 3

29

Fig. 4

30

Fig. 5

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Tables Table 1 The degradation of OTC, byproducts, bacterial abundance and diversity in different treatment. ADOTC

OTC residual

EOTC ratio

CK

41.92±0.78a

7.23±0.14a

0.49±0.001b

7.01±0.81b

8.83±0.18c

E

33.31±0.31c

7.00±0.12ab

0.73±0.05a

9.46±0.01a

9.10±0.08a

AM

35.06±0.56b

4.79±0.16c

0.71±0.01a

9.65±0.13a

8.86±0.02bc

27.82±0.43d

6.82±0.12b

0.52±0.10b

9.62±0.06a

9.06±0.10ab

E+AM

ratio

16s abundance

Shannon index

a

Significance due to E

***

***

NS

**

*

AM

***

***

NS

***

NS

E*AM

*

***

***

**

NS

Data are the means of three replicates ±SD and were compared by Duncan's multiple range tests. Within each column (3 values) the values with the same lower case letter are not significantly different at p<0.05. NS not significant; **P < 0.01, *P < 0.05. a By analysis of variance.

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Table 2 Pearson’s correlation coefficients relating OTC and its productions and the relative abundance of bacterial groups at the class, family, and genus level, respectively.

Classification level Class

Family

Genus

Pearson's correlation coefficients Cytophagia (r2=-0.784**) OTC Anaerolineae (r2=-0.706*) OTC Flavobacteriia (r2=-0.817**) OTC Gammaproteobacteria (r2=-0.583*) EOTC Planctomycetia (r2=-0.661*) EOTC Pirellulaceae (r2=-0.799**) EOTC Cytophagaceae (r2=-0.776**) OTC Flavobacteriaceae (r2=-0830**) OTC Streptomycetaceae (r2=0.863**) OTC Alteromonadaceae (r2=-0.675*) OTC Xanthomonadaceae (r2=0.872**) OTC Pseudomonas (r2=-0.632*) OTC Cellvibrio (r2=-0.765**) OTC Glycomyces (r2=-0.709**) EOTC Nonomuraea (r2=0.670*) OTC (r2=-0.601*) EOTC Bacterial group

*p<0.05; **p<0.01.

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