The performance of biochar-microbe multiple biochemical material on bioremediation and soil micro-ecology in the cadmium aged soil

Science of the Total Environment 686 (2019) 719–728

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Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv

The performance of biochar-microbe multiple biochemical material on bioremediation and soil micro-ecology in the cadmium aged soil Bin Wu a, Ziru Wang a, Yuxing Zhao a, Yuanming Gu a, Ying Wang a, Jiang Yu b,⁎, Heng Xu a,⁎ a b

Key Laboratory of Bio-Resource and Eco-Evironment of Ministry of Education, College of Life Sciences, Sichuan University, Chengdu 610065, PR China College of Architecture and environment, Sichuan University, Cheng du 610065,PR China

H I G H L I G H T S

G R A P H I C A L

A B S T R A C T

• The multiple biochemical material was prepared using SNB6 with biochar. • The HOAc-extractable Cd significantly increased by biochemical material. • Bioremediation efficiency was significantly enhanced by the biochemical material. • Soil micro-ecology was improved by biochemical material and accumulator. • The SNB6 was successfully colonized in rhizosphere interface.

a r t i c l e

i n f o

Article history: Received 10 May 2019 Received in revised form 3 June 2019 Accepted 3 June 2019 Available online 04 June 2019 Editor: Frederic Coulon Keywords: Biochemical material Bioremediation Cadmium Micro-ecology Microbial community

a b s t r a c t Biochar (BC) and plant growth promoting bacteria (PGPR) have been widely applied to improve the qualities of heavy metal contaminated soil, while the synergy effect of BC and PGPR on the bioremediation of cadmium (Cd) contaminated soil was less studied. In this study, a novel PGPR strain SNB6 was isolated and then immobilized on BC as the multiple biochemical material (BCM) as well as combined with vetiver grass (Chrysopogon zizanioides L.) to form BC-PGPR-accumulator system. The promoting effects of BCM on bioremediation and soil microbiology were comprehensively investigated. SEM and FTIR analysis indicated that the strain SNB6 was successfully fixed on BC and the functional groups between BC and SNB6 surface contributed to the immobilization effect. The BCM significantly enhanced the Cd content and bioaccumulation factor (BCF) of accumulator, about 412.35% and 403.41% higher than that of control, respectively. Meanwhile, the biomass of fresh and dry accumulator in the BCM treatment was 227.27% and 178.33% higher than that of control. In addition, the system significantly increased the proportion of HOAc-extractable Cd and soil micro-ecology. Microbial counts and soil enzyme activities in rhizosphere were both significantly improved by the interaction of BCM and C. zizanioides. Furthermore, the strain SNB6 in the rhizosphere interface was successfully colonized, and soil microbial community was evaluated to understand the microbial diversity after bioremediation. Our study indicated that the BCM could significantly enhance the bioremediation efficiency and drive the soil micro-ecology, and the BC-PGPRaccumulator system provided a feasible pathway to remediate heavy metal contaminated sites. © 2019 Published by Elsevier B.V.

⁎ Corresponding authors at: Sichuan University, No. 24 South Section 1, Yihuan Road, Chengdu 610065, PR China. E-mail addresses: [email protected] (J. Yu), [email protected] (H. Xu).

https://doi.org/10.1016/j.scitotenv.2019.06.041 0048-9697/© 2019 Published by Elsevier B.V.

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1. Introduction Cadmium (Cd) is a toxic contaminant, which has caused a serious environmental problem due to the wastewater irrigation, chemical fertilizer application and rapid industrialization (Mahar et al., 2016). In China, approximately 2.78 × 109 m2 of agriculture soil were polluted with Cd (Liu et al., 2015a). Thereby, it is urgent to develop novel materials and effective technologies to remediate Cd contaminated soil. Bioremediation technology by hyperaccumulators is one of promising methods to remove Cd from soil with low cost and environmental friendly (Ali et al., 2013). Among the various hyperaccumulators, vetiver grass (Chrysopogon zizanioides L.) has the advantages of tall (1–2 m), fast growing, easily cultivation, with a long root system (3–4 m) that are potential for the bioremediation of Cd contaminated soil (Pidatala et al., 2016). In addition, C. zizanioides can be grown in saline, lime, peat, low fertile soil conditions, and even in extreme temperatures (Attinti et al., 2017). As a versatile plant in agriculture, many studies have been reported for its effectiveness on the use of remediation of heavy metal, petroleum and 137Cs (Chantachon et al., 2004; Roongtanakiat and Akharawutchayanon, 2017; Yang et al., 2003). Compared with other hyperaccumulators, the more developed root system can penetrate deeper layers of soil, which is favor of cleaning up contaminants of deep soil. However, in highly Cd contaminated soil, the Cd toxicity can inhibit root growth and cell division, consequently resulting in inhibiting plant growth and cadmium uptake (Wang et al., 2007). Therefore, it is necessary to develop novel technologies to improve the bioremediation efficiency, especially in highly contaminated soil. Many researches have been focused on increasing the Cd mobilization and promoting the plant growth to enhance bioremediation efficiency by the utilization of indigenous microorganisms (Lebeau et al., 2008). Soil is one of largest microbial libraries on earth and distributed large of microbial diversity with an estimated 107–109 distinct bacterial species, which possess vital effects to regulate soil ecological functions (Wu et al., 2018a). Indeed, some metal-resistant bacteria, such as plant growth-promoting bacteria (PGPR), could interact with hyperaccumulators through a series of physiological and biochemical activities including of exudation of extracellular substances such as indole-3-acetic acid (IAA), siderophore, biosurfactant and 1aminocycloppropane-1-carboxylic acid (ACC) deaminase to increase the biomass of accumulator and the Cd accumulation in plant tissue (Wang et al., 2017 Prapagdee et al., 2013). However, there is lacking of the studies regarding the microbe-C. zizanioides model on the removal of Cd compared with other microbe-plant researches. Moreover, almost all of these researches were conducted by the competition indigenous

Table 1 The PGP traits of PGPR. Bacteria

Phosphate solubilizinga (mg/L)

Siderophore productiona (A/Ar)

IAA productiona (mg/L)

SSB1 FCB2 HBK3 CSB4 THB5 SNB6 CCF7

53.29 ± 4.48 28.47 ± 2.59 19.14 ± 2.67 86.37 ± 5.16 26.75 ± 1.88 127.25 ± 7.24 57.43 ± 2.54

0.71 ± 0.14 0.43 ± 0.08 0.82 ± 0.17 0.64 ± 0.12 0.84 ± 0.14 0.47 ± 0.07 0.53 ± 0.23

21.18 ± 1.38 14.32 ± 1.27 8.75 ± 0.84 10.76 ± 1.31 16.54 ± 1.16 22.09 ± 1.04 18.44 ± 1.42

a

Values are expressed as mean ± standard error (n = 3).

microorganisms without sheltering. The direct incubation of inoculum would reduce the ability to colonize and reduce its vertical transport in soil. Therefore, a suitable inoculum material should be considered to better utilization for the microbe-hyperaccumulator technology. Biochar (BC) is a kind of carbon-enriched and porous chemical material, which has been proposed as an approach to improve soil quality (Wu et al., 2016). The BC can be shelter due to its pore structure that provides more habitable for microbes than soil does, thus ensure the quality of inoculum (Quilliam et al., 2013a). Moreover, BC contains plentiful nutrients (K, Mg, N and P etc.) and can enrich soil nutrients (Rodríguez-Vila et al., 2016). Some reports indicated that BC can be as slow-release fertilizer, bringing long-term benefits for the growth of microorganisms and plants (Masiello et al., 2013). Thus, we inferred that PGPR immobilized by BC as the biochemical material (BCM) could be an innovative idea to enhance of the efficiency of bioremediation. Therefore, we designed a novel system for the bioremediation of Cd contaminated soil by using BC as shelter and slow-release fertilizer for PGPR, the PGPR to activate Cd and promote accumulator growth, C. zizanioides as accumulator to accumulate Cd from soil. In this study, the Cd tolerance and PGP traits of PGPR were analyzed. The morphological structure and functional groups of BC, PGPR and BCM were characterized by scanning electron microscope (SEM) and fourier transform infrared spectra (FTIR). In addition, the effects of BCM on bioremediation efficiency and soil micro-biology were comprehensively investigated. 2. Material and methods 2.1. Materials The aged soil was collected from the campus of Sichuan University, Chengdu, China, where a remote experimental site has been applied for scientific research about heavy metals for many years. Up to now,

Fig. 1. The growth curves of isolated PGPR under initial concentration of 20 mg/L Cd (a) and effect of Cd concentrations on the growth of the strain SNB6 (b). Error bars represent standard deviations, and means with different letters are significantly different from each other (P b 0.05) according to the LSD test (n = 3).

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much works have been done and part of the soil used for experiments was highly contaminated with heavy metals. The soil used in this study was highly contaminated with Cd due to the previous study (Wu et al., 2018a) and the main characteristics were shown in Table S1. The seeds of C. zizanioides were offered by the academy of agricultural science of Jiangsu province. All regents used in this study were analytical regents and purchased from the Kelong Chemical Reagent and Sigma–Aldrich companies. Deionized (DI) water (18.2 MΩ∙cm) was used to prepare all chemicals. 2.1.1. Isolation and characterization of PGPR The PGPR was isolated from the rhizosphere soil of C. zizanioides. The growth capacity under Cd stress was verified in lysogeny broth (LB) media with the increasing concentrations of Cd (0–150 mg/L) at the conditions of 37 °C and 160 rpm. The production of IAA, siderophore, and phosphate-solubilizing substances as PGP trains was determined as described by Bric et al. (1991), Schwyn and Neilands (1987), Fiske and Subbarow (1925), respectively. According to the Cd tolerance and PGP traits, the PGPR strain marked as SNB6 was selected for the following study. The strain SNB6 identification was carried out by 16S rDNA sequencing as previous method and the similar DNA sequences were matched in GeneBank database using BLAST (Byers et al., 1998). Then, the phylogenetic tree of strain SNB6 was constructed using the MEGA 5.0 program. 2.1.2. Preparation and characterization of BCM BC used in this study was obtained from waste wheat straw (Hordeum vulgare L.) products (Foshan, China) and the preparation method was ascribed in the previous study (Wu et al., 2016). After that, BC was washed carefully with DI water until the neutral pH was achieved. To prepare BCM material, the immobilization of SNB6 was conducted in the optimal condition, which was ascribed in our previous study (Xiao et al., 2017). Briefly, the 100 mL SNB6 suspension of logarithmic phrase and 5 g BC were mixed with a ratio of 20:1 (v:w) and shaked for 12 h at 160 rpm. Both non-immobilized and immobilized SNB6 were rinsed with DI water and stored for use. To observe the structure and the functional groups, the non-immobilized and immobilized SNB6 were fixed with 2.5% glutaraldehyde at 4 °C overnight, then washed three times with phosphate buffer solution, and dehydrated serially with different concentrations of ethanol. Finally, the samples were dried with critical point drying method and the structure was observed by SEM (JSM-7500F). The surface functional groups of samples were evaluated by FTIR (Nicolet 6700). 2.2. Experimental design The plastic pots containing 2 kg Cd contaminated soil were used for this experiment. The germination of C. zizanioides seeds was conducted in a darkness condition for three days. Subsequently, bacterial suspension, BC and BCM were added into pots, respectively, and then mixed thoroughly with soil. The amount of these materials added were 20 seeds, 100 mL bacteria suspension (~108) and 100 mL BCM suspension. After that, the healthy and uniform sprouts of C. zizanioides were selected and planted in the above soil. The treatments in the amendment factors were T1 (CK), T2 (P), T3 (BC), T4 (BC P), T5 (B), T6 (B P), T7 (BCM), T8 (BCM P) and each treatment was conducted in three replicates. “CK”, “P”, “B” and “BCM” represent control without treatment, soil planted with C. zizanioides, soil incubated with bacterial suspension, soil incubated with BCM suspension, respectively. During the period of incubation, all treatments were watered each day with DI water to keep stable water holding capability, and the temperature range was 25–28 °C during day and 18–22 °C during night. After four weeks of incubation, the C. zizanioides was harvested, washed with DI carefully and the fresh weight was recorded. Then, the C. zizanioides was dried at 60 °C for 24 h, and the dry weight was recorded. Soil adhering to the roots after shaking was defined as rhizosphere soil (Baudoin et al.,

Fig. 2. SEM images of BC (a) the strain Serratia sp. SNB6 (b) and BCM (c).

2003). After gently shaking the accumulator roots, the rhizosphere soil, free of roots, was carefully collected per treatment. Half of fresh rhizosphere soil was used to determine biochemical properties, and half of rhizosphere soil was frozen with liquid nitrogen and then stored at −80 °C to analyze microbial diversity. 2.3. Heavy metal analysis The powered C. zizanioides (0.1 g) was digested with the mixed solution of HNO3, HClO4 and HF (5:4:3, v/v) and the Cd content was

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Fig. 5. Different fractions of Cd in different treatments. Fig. 3. FTIR spectra of BC, the strain Serratia sp. SNB6 and BCM.

measured by atomic absorption spectroscopy (AAS; VARIAN, SpecterAA-220Fs) (Wu et al., 2016). Metal fractions were extracted to evaluate the metal bioavailability and transformation by BCR procedure (Kartal et al., 2006; Wu et al., 2018a). Briefly, 1 g of rhizosphere soil was shaken with 40 mL of 0.11 M CH3COOH at 25 °C, 250 rpm for 16 h to assay the content of acetic acid extraction state. Then, the above residue was shaken with a 40 mL mixture of 0.5 M NH2OH·HCl and 0.05 M HNO3 at 25 °C, 250 rpm for 16 h to assay the state in combination with oxidation. Subsequently, the abovementioned residue was heated with 10 mL 30% H2O2 (pH = 2.5, 85 °C) in a water bath until the liquid b1 mL, and then extracted with 50 mL 1.0 M CH3COONH4 (pH = 2) at 25 °C, 250 rpm for 16 h to assay the state in combination with reduction. Finally, the abovementioned residual soil (0.1 g) was digested with mixed solution of HNO3, HClO4 and HF (5:4:3, v/v) using a microwave digestion method to extract the residual fraction. Thus, the method defined metal into four fractions: HOAc extractable, reducible, oxidizable and residual fractions. 2.4. Soil micro-ecology analysis The soil was carefully collected from plant rhizosphere and used to micro-ecological analysis. The microbial biomass and enzyme activities

Fig. 4. Plant biomass in different treatments. Error bars represent standard deviations, and means with different letters are significantly different from each other (P b 0.05) according to the LSD test (n = 3).

were determined to reflect soil biochemical quality. The bacterial and fungi counts were recorded by the plate spread method as described by Vieira and Nahas (2005) and the number of microbes was defined as log CFU/g soil. Dehydrogenase activity was evaluated by triphenylformazan (TPF) at the wavelength of 492 nm and expressed as μg TPF/(g soil·24 h) (Benefield et al., 1977). Urease activity was assayed by an NH4 coloured complex at the wavelength of 578 nm and expressed as μmol NH4 /(g soil·24 h) (Yan et al., 2013). Fluorescein diacetate (FDA) hydrolysis activity was determined by at the wavelength of 490 nm and expressed as fluorescein μg/(g soil·h) (Green et al., 2006). Acid phosphatase activity was measured on the basis of p-nitrophenol (pNP) release at the wavelength of 400 nm and expressed as μg pNP/(g soil·h) (Aarle and Plassard, 2010). Invertase activity was determined by amount of glucose at the wavelength of 508 nm and expressed as μg glucose/(g soil·24 h) (Gu et al., 2009).

2.5. Microbial diversity analysis The bacterial DNA of rhizosphere soil was extracted using a Soil DNA Kit (Omega Bio-tek, Norcross, GA, U.S.) according to manufacturer's protocols, and then the 16S rRNA gene was amplified with primers 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGAC TACHVGGGTWTCTAAT-3′) by thermocycler PCR system (GeneAmp 9700, ABI, USA). The bacterial diversity was analyzed on an Illumina

Fig. 6. Cd accumulation in plant and BCF values in different treatments. Error bars represent standard deviations, and means with different letters are significantly different from each other (P b 0.05) according to the LSD test (n = 3).

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MiSeq platform (Illumina, San Diego, USA) according to the standard protocols by Majorbio Bio-Pharm Technology Co. Ltd. (Shanghai, China).

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3. Results and discussion 3.1. Characterization of biochemical materials

2.6. Data analysis In this study, all treatments were performed by three replicates. Bioaccumulation factors (BCF) were calculated according to the following formula:

BCF ¼

Metal concentration in plant Metal concentration in soil:

Statistical significance was performed using SPSS 18.0 package and statistics were performed using the Origin 8.0 (USA).

3.1.1. Metal resistance and PGP trains of PGPR In this study, seven PGPR strains were isolated from rhizosphere soil. The capability of Cd resistance for isolates was determined at the Cd concentration of 20 mg/L and the result was showed in Fig. 1. Among these strains, the strain SNB6 grow better and the value of OD600nm was up to 1.81 within 24 h (Fig. 1a), which indicated that the strain SNB6 possessed the strongest living capability to resist Cd toxicity. It was observed that the growth of the strain SNB6 was varied based on the different Cd concentrations in the growth medium (Fig. 1b). Among these treatments, the maximum bacterial growth was observed at control (without Cd addition) and slightly decreased at the

Fig. 7. Microbial numbers (a) and enzyme activities (b-f) of soil in different treatments. Error bars represent standard deviations, and means with different letters are significantly different from each other (P b 0.05) according to the LSD test (n = 3).

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a

Percent of community abundance on Genus level

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Community barplot analysis 1

0.75

0.5

0.25

0 T1

T2

T4

T6

T8

Samples Nocardioides Lysobacter unclassified_f__Nocardioidaceae norank_o__JG30-KF-CM45 Sphingomonas unclassified_o__Sphingomonadales Serratia Knoellia norank_f__Cytophagaceae Marmoricola norank_c__Gemmatimonadetes norank_p__Saccharibacteria norank_o__AKYG1722 norank_o__Acidimicrobiales Methylophaga Bacillus norank_c__Acidobacteria norank_c__Gitt-GS-136 Limnobacter unclassified_f__Micrococcaceae norank_f__Anaerolineaceae norank_c__KD4-96 Arthrobacter Arenimonas others

b

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Fig. 8. Relative abundance (a), community heatmap (b) and circos (c) of bacterial diversity in rhizosphere soil with different treatments.

concentrations 20 and 50 mg/L Cd, while the growth was inhibited at the concentrations 100 and 150 mg/L Cd. Similar with other studies, although the metal resistant strains could have tolerance toward the toxicity of heavy metal, exceed concentrations of heavy metal could inhibit their metabolism, thus slowing down their growth (Huang et al., 2014; Khan et al., 2016). Nevertheless, compared with other metal resistant strains, the growth status of the strain SNB6 was better under Cd stress (Sheng et al., 2016; Yao et al., 2016). Meanwhile, the PGP traits (phosphate solubilizing, IAA and siderophore production) of these PGPR were measured and the results showed in Table 1. In our study, the phosphate solubilizing capacity of isolates was at the levels of 19.14–127.15 mg/L and the strain SNB6 showed the highest phosphate solubilizing capacity. In addition, these isolates could produce IAA at the levels of 8.75–22.09 mg/L, especially for the strain SSB1 and strain SNB6 whose production over 20 mg/L. Moreover, the results showed that the strain SNB6 could secret more siderophores than other isolates. Therefore, the strain SNB6 possessed

the preferable Cd tolerance and PGP traits. Moreover, compared with other reports, these productions of the strain SNB6 were relative higher than those produced by some of other PGPR (Han et al., 2017; Prapagdee et al., 2013; Ying et al., 2016b). Based on the capabilities of metal tolerance and PGP traits, the strain SNB6 was selected for further study. The 16S rDNA sequencing of the strain SNB6 was measured and listed in Table S2. According to the match of similar DNA sequences in NCBI GeneBank, the phylogenetic tree illustrated that the strain SNB6 was highly similar with Serratia marcescens strain (Fig. S1). 3.1.2. Characterization of the BCM The main characteristics of BC and BCM were presented in Table S3. BC contained high organic matter that could supply nutrients for the survival of the strain SNB6 (Zhu et al., 2017). The structures of BC, the strain SNB6 and BCM were presented in Fig. 2. The structure of BC surface was polyporous, which provided a

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suitable shelter for the strain SNB6 (Xiao et al., 2017). Moreover, the strain SNB6 was successfully immobilized in BC. The living cells of the strain SNB6 attached in BC could protect them from the direct toxicity of Cd and provide the habitat for colonization (Quilliam et al., 2013b). In addition, FTIR spectra illustrated the possible interaction between the strain SNB6 and BC (Fig. 3). The broad band around 3419 cm−1 corresponds to the –OH groups (Tang et al., 2014). The peaks at about 2935 cm−1 and 1095 cm −1 were related O\\H and C\\O bending vibration, respectively (Wu et al., 2018b). The appearance of peaks with increased elongation and intensity in BCM showed massive functional groups enriched in the surface contributed to bounding the strain SNB6.

3.2. The growth response of C. zizanioides The fresh and dry weights of C. zizanioides were presented in Fig. 4, which showed the effect of different materials on the growth response of C. zizanioides. Compared with the untreated group, the addition of materials showed positive effect on the growth of C. zizanioides. The biomass of C. zizanioides in BC treatment slightly increased, whose fresh and dry weights were 123.31% and 117.46% of control, respectively. Nevertheless, the biomass of C. zizanioides was promoted mostly by the BCM that fresh and dry weights were 227.27% and 178.33% of control, respectively. These results were related to the PGP traits of the strain SNB6 that could produce IAA and siderophore and dissolve inorganic phosphorus (Table 1). It was reported that IAA-producing bacteria could stimulate root elongation and plant growth in heavy metal contaminated soil (Ying et al., 2016b), while siderophore could reduce the ethylene levels and protect the roots from the stress of heavy metal (Ying et al., 2016a). The phosphate solubilizing by microbes could transfer the inorganic phosphate to available phosphate, thus promoting the growth of C. zizanioides (Rajkumar and Freitas, 2008). The BC provided the nutrients and shelter for SNB6 that facilitated SNB6 secret more compounds of promoting plant growth. Therefore, the utilization of BCM could increase biomass of plant, sequentially enhance the potential of bioremediation efficiency.

3.3. Fractions and transformation of Cd in soil The fractions and transformation of Cd were determined and the results were showed in Fig. 5. Compared with control, the HOAcextractable Cd decreased 18.89% in T2 group, which indicated that C. zizanioides could effectively extract the available Cd from soil. Nevertheless, it was observed that HOAc-extractable Cd increased after incubation with SNB6 and BCM. The maximum percentage (44.70%) of HOAc-extractable Cd was observed in T8 group that showed the BCM could effectively activate Cd in soil. These results could be explained from two aspects. On the one hand, the SNB6 could produce acid and surfactant substances during the incubation period, thus contributing to the desorption of Cd from soil (Gao et al., 2004). On the other hand, the SNB6 immobilized in BC occupied functional groups of BC, which competitively restrained the binding of Cd with functional groups. Meanwhile, the oxidizable and reducible Cd were slightly decreased in SNB6 and BCM groups, which suggested the increase of HOAcextractable Cd was result from the transformation of oxidizable and reducible Cd (Liu et al., 2015b). Metal bioavailability was a crucial important factor of the bioremediation and the metal fractions had varied mobilization behaviors under the effect of different functional microbes (Abhilash et al., 2014; Thierry et al., 2008). Recently, many studies have indicated that PGPR with high metal resistance could mobilize heavy metal through the secretion of metal chelators (e.g. siderophores, biosurfactants and organic acid) and phosphate solubilization (Karimzadeh et al., 2012; Mani et al., 2010; Salamone et al., 2001).

3.4. The Cd content in accumulator To evaluate the bioaccumulation effect of Cd, the Cd content of C. zizanioides and the BCF values were evaluated in Fig. 6. The content of Cd was 13.08–52.55 mg/kg among the C. zizanioides planted-groups and the maximum value was observed in presence of BCM. In the T6 and T8 groups, the accumulation of Cd was 259.47% and 412.35% higher than that of control. Moreover, the BCF values significantly increased from 0.88 (T2) to 2.42 (T6) and 3.55 (T8), respectively. These results revealed that the strong capacity of C. zizanioides in enriching Cd from soil, while the single SNB6 and BCM could both significantly promote the accumulation efficiency of Cd, which was resulted from the Cd bioavailability increased after the incubation of SNB6 and BCM (Fig. 5). The BC assisted with SNB6 simultaneously relieved the Cd toxicity for C. zizanioides, thus strengthening the removal efficiency of Cd from heavy contaminated soil. 3.5. Soil micro-ecology in rhizosphere interface To evaluate the bioremediation effect of this system on soil microecology, the total number of bacteria and fungi as well as the activities of five important enzymes were determined (Fig. 7). It was observed that both the counts of bacteria and fungi increased in all treatments, which were approximately 100.77–119.42% and 102.93–123.29% of control (Fig. 7a). The microbial numbers slightly increased in BC groups and significantly increased in groups incubated with SNB6 and BCM. In addition, the microbial numbers in accumulator groups (log 6.02–6.90 CFU/g soil for bacteria and log 3.50–4.19 CFU/g soil for fungi) were higher than non-accumulator groups (log 5.78–6.71 CFU/g soil for bacteria and log 3.40–4.12 CFU/g soil for fungi). The maximum values of bacterial and fungal numbers were observed in T8 and T6 groups, respectively, about 119.32% and 132.28% higher than that of control. Soil enzyme activities were presented in Fig. 7b-f, which showed that the activities of dehydrogenase, FDA hydrolase, urease, acid phosphate and invertase almost increased in different treatments. Dehydrogenase activity increased by 294.57–595.18% in all treatments compared with control. The incubation of single BCM and BCMaccumulator significantly increased the dehydrogenase activity, which was approximately 467.34% and 514.17% of control, respectively. In the presence of SNB6 with C. zizanioides, dehydrogenase activity was reach to maximum value, about 594.82% higher than that of control. Except of T3 group, FDA hydrolase activity increased in different treatments, about 13.49–159.89% higher than that of control. In the T5 group, FDA hydrolase activity reached to maximum, about 258.64% higher than that of control. Urease activity was raised by 108.12–182.13% of control and reached to maximum in the presence of BCM-accumulator. In the treatment of SNB6 with C. zizanioides, the acid phosphate activity significantly increased, about 132.12% of control, whereas it not significantly varied in other treatments. In addition, the invertase activity increased in different treatments, about 103.97–132.14% of control. In the treatment of BCM, the maximum value of invertase activity was observed (about 22.80 mg glucose/g soil/24 h). The microbial counts and enzyme activities as important biochemical indexes have generally been used to reflect the micro-ecology quality of soil (Bandick and Dick, 1999; Li et al., 2009). Overall, our results indicated that these biochemical properties presented above has been improved by either single or multiple combination of these materials. Among these different treatments, SNB6 and BCM incubation significantly increased the microbial number and enzyme activities, especially for dehydrogenase, urease and invertase. After incubation of accumulator, dehydrogenase, FDA hydrolase, urease and acid phosphate variously increased in different treatments. These results could be explained from two aspects. On the one hand, the metabolites (organic acid, thiol, protein and available phosphate, etc.) produced by the

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interaction of accumulator and SNB6 would strengthen the soil microbial activity (Epelde et al., 2009). On the other hand, the BC stored a range of nutrients and acted as slow-release fertilizer, contributing to the growth of microbes and the secretion of enzymes (Zhu et al., 2017). Moreover, the BC provided the shelter for SNB6, which would further contribute the positive effect on SNB6 against the Cd toxicity (Quilliam et al., 2013b). Therefore, these results illustrated that the application of the BC-PGPR-accumulator system could notably improve the biochemical quality of soil micro-ecology in Cd contaminated soil. 3.6. Microbial diversity in rhizosphere interface The colonization of SNB6 and the microbial diversity in rhizosphere interface were analyzed by high-throughput sequencing Fig. 8. Fig. 8a illustrated that microbial community was significantly influenced by different treatments. It was observed that Serratia sp. significantly increased in T6 (0.65%) and T8 (13.11%) groups compared with control (abundance b0.5%), which indicated that the strain SNB6 was successfully colonized in soil and could interactive with C. zizanioides. In the BCM treatment, the relative abundance of Serratia sp. significantly increased, which suggested that the shelter of BC could be conducive to SNB6 colonization and played positive correlation with higher Cd removal capacity. The Serratia sp. was proved to efficiently enhance the heavy metal resistance of accumulator and promote its growth (Han et al., 2017; Xiao et al., 2017). Community heatmap showed the similarity and difference of these groups at the genus level (Fig. 8b). Cluster analysis showed a similar bacterial diversity in accumulator planted groups. In addition, Circus analysis visually reflected that the dominant genus in different groups (Fig. 8c), which showed similar trend with relative abundance. It was observed that Serratia sp. was one of dominant genus and its percentage reached to 94% in BCMaccumulator group. Meanwhile, in the BCM treatment, the Alphadiversity indexes slightly decreased compared with other groups (Table S4), which might be related to the competition of the strain SNB6 with indigenous microorganisms causing the inferior population decrease. These results fully showed that the immobilization of the strain SNB6 by BC could be beneficial to the colonization of the strain SNB6 in the rhizosphere soil. The more colonization of the strain SNB6 implied better effect of Cd mobilization and C. zizanioides growth, thus significantly improving the Cd removal efficiency. 4. Conclusions This study indicated the application of the BCM multiple biochemical materials could effectively enhance Cd accumulation of C. zizanioides and improve the soil biochemical qualities in Cd highly contaminated soil. The BCM significantly increased the HOAc-extractable Cd and C. zizanioides biomass as well as Cd accumulation. In addition, the soil microbial counts and enzyme activities significantly increased in the incubation of BCM-accumulator. The combination of BC-PGPRaccumulator system provided a low-cost and effective route to remediate Cd contaminated soil. Apart from soil remediation, the idea of developing novel biochemical materials by utilizing BC and PGPR could facilitate the progress in agriculture, such as assisting plant growth and inhibiting plant disease. Acknowledgements This study was financially supported by the National Key Research and Development Program (2018YFC1802605), Agricultural Science and Technology Achievements Transformation Program of Sichuan Province (2017NZZJ008), the Key Research and Development Program of Sichuan Province (2017SZ0181), the Science and Technology Supportive Project of Sichuan Province (2018RZ0110). The authors also wish to thank Professor Guanglei Cheng and Dong Yu from Sichuan University for the technical assistance.

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