International Journal of Phytoremediation

ISSN: 1522-6514 (Print) 1549-7879 (Online) Journal homepage: http://www.tandfonline.com/loi/bijp20

Enhanced phytoremediation of soils contaminated

with PAHs by arbuscular mycorrhiza and
rhizobium

Cheng-Gang Ren, Cun-Cui Kong, Bian bian, Wei Liu, Yan Li, Yong-Ming Luo &
Xie Zhi-Hong

To cite this article: Cheng-Gang Ren, Cun-Cui Kong, Bian bian, Wei Liu, Yan Li, Yong-Ming
Luo & Xie Zhi-Hong (2017): Enhanced phytoremediation of soils contaminated with PAHs
by arbuscular mycorrhiza and rhizobium, International Journal of Phytoremediation, DOI:
10.1080/15226514.2017.1284755

To link to this article: http://dx.doi.org/10.1080/15226514.2017.1284755

Accepted author version posted online: 06

Feb 2017.

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Enhanced phytoremediation of soils contaminated with PAHs by arbuscular mycorrhiza and

rhizobium

Cheng-Gang Ren1; Cun-Cui Kong1,3; Bian bian1; Wei Liu1; Yan Li1; Yong-Ming Luo2;

Zhi-Hong Xie1,*

1
Key Laboratory of Biology and Utilization of Biological Resources of Coastal Zone, Yantai

Institute of Coastal Zone Research, Chinese Academy of Sciences, Yantai 264003, China

2
Key Laboratory of Coastal Environmental Processes and Ecological Remediation, Yantai

Institute of Coastal Zone Research, Chinese Academy of Sciences, Yantai 264003, China

3
Shanxi Agricultural University, Taigu 030801, China
*
Corresponding author: Zhi-Hong Xie, Email: zhxie@yic.ac.cn, Telephone:

+86-0535-2109183

Cheng-Gang Ren and Cun-Cui Kong contributed equally to this study.

Abstract:

Greenhouse experiment was conducted to evaluate the potential effectiveness of a legume

(Sesbania cannabina), arbuscular mycorrhizal fungi (AMF) (Glomus mosseae) and

rhizobia (Ensifer sp.) symbiosis for remediation of PAHs in spiked soil. AMF and

rhizobia had a beneficial impact on each other in the triple symbiosis. AMF and/or

rhizobia significantly increased plant biomass and PAHs accumulation in plants. The

highest PAHs dissipation was observed in plant + AMF + rhizobia treated soil, in which

>97 and 85-87% of phenanthrene and pyrene, respectively, had been degraded, whereas

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81-85% and 72-75% had been degraded in plant-treated soil. During the experiment, a

relatively large amount of water-soluble phenolic compounds were detected in soils of

AMF and/or rhizobia treatment. It matches well with the high microbial activity and soil

enzymes activity. These results suggest that the mutual interactions in the triple

symbiosis enhanced PAHs degradation via stimulating both microbial development and

soil enzyme activity. The mutual interactions between rhizobia and AMF help to improve

phytoremediation efficiency of PAHs by S. cannabina.

Keywords:

AMF; Biotoremediation; Polycyclic aromatic hydrocarbons; Rhizobia; Sesbania cannabina

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1 Introduction

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous as pollutants from petroleum and coal

utilization and are of great environmental concerns because of their strong carcinogenicity,

teratogenicity and ecotoxicity. Soils have become an important environmental sink for PAHs

because of their large holding capacity for pollutants. Among the various types of bioremediation

strategies for such polluted soils, phytoremediation represents an environmentally friendly and

cost effective approach. Phytoremediation also provides intangible benefits for the soil

ecosystem, including soil carbon equestration, soil improvement, biomass and bio-fuel

production, and biodiversity maintenance (Rajkumar et al. 2012).

Legumes are plants that play essential roles in nitrogen cycling due to their symbiosis with the

nitrogen-fixing bacteria. Many reports have noted that some leguminous species are heavy-metal

resistant and can significantly promote the dissipation of organic pollutants, such as PAHs,

polychlorinated biphenyls (PCBs) (Hamdi et al. 2012; Li et al. 2013). Sesbania cannabina

(Retzius) Poir plant can tolerates adverse environment including organic contaminated soil and it

has been successfully used in remediation of petroleum-polluted soils (Farhana et al. 2012).

Rhizobia have been studied in relation to phytoremediation because of their potential to form

symbiotic associations with leguminous plants. Rhizobia colonize the roots of legumes where

they fix atmospheric N2, which can be utilized for plant growth. Rhizobia are found in

contaminated environments where various toxic chemicals are present (Yang et al. 2005).

Several bacterial species in the genus Rhizobium are able to utilize PAHs, PCBs, or heterocyclic

aromatic compounds (Johnson et al. 2005; Xu et al. 2010).

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Arbuscular mycorrhizal fungi (AMF), a group of ubiquitous soil-borne fungi, have positive

impacts on plant establishment and survival in contaminated soils by increasing nutrient uptake,

improving drought tolerance. In a biodegradation context, AM fungi are obligate symbionts with

limited capacity for degradation of organic pollutants (Michelsen et al. 1998). AMF do,

however, interact with and modify the microbial communities that the hyphae encounter in soil,

and in this manner they can affect microbial degradation processes indirectly (Joner et al. 2003).

AMF play positive roles on the remediation of PAHs-contaminated soils (Yu et al. 2011; Aranda

et al. 2013), and affect the uptake and translocation of PAHs in plants (Verdin et al., 2006).

Thus, the symbiotic association between S. cannabina, rhizobia and AMF might be exploited for

remediation of contaminated soils. Previous studies have used the legume (e.g. alfalfa) -

rhizobium symbiosis to remediate soils contaminated with persistent organic pollutants such as

PCBs and petroleum hydrocarbon compounds (PHC) with good outcomes (Mehmannavaz et al.

2002; Radwan et al. 2007).

However, the interaction between S. cannabina, rhizobia and AMF as well as their effects on

PAHs dissipation are still unknown. The two aims of the present study were: (1) to investigate

the potential of the S. cannabina, rhizobia and AMF associations to remediate 3-and 4-ring

PAHs-contaminated soils, and (2) to study the related changes in soil PAHs-degrading microbial

activity and enzyme activities.

2 Materials and methods

2.1 Preparation of PHE and PYR contaminated soil

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Soil was collected from the campus of Yantai Institute of Coastal Zone Research, Shandong,

China, with undetectable phenanthrene (PHE) and 12 mg kg1 pyrene (PYR). The total

concentration of 16 US EPA priority PAHs was 34 mg kg1 dry soil, with the concentrations of

2-, 3-, 4-, 5- and 6-ring PAHs of 0, 0, 12, 15 and 7 mg kg1, respectively. This concentration (34

mg kg1) was below the threshold level required for Chinese soil PAHs quality (45 mg kg1 dry

weight) for residential use (GB15618-2008) (E.P.A. China, 2008). The soil was air-dried and

passed through 2mm sieve to remove stones and roots. The particle size distribution (52.1%

sand, 27% silt, and 20.9% clay) identified the soil as a sandy loam soil, with a pH of 5.62, 2.03%

soil organic matter. The cation exchange capacity (CEC) was 9.96 c mol kg1 and electrical

conductivity (EC) was 254.5 ms cm1. The nutrient levels were18.5 mg kg1 NO3-N, 21.3 mg

kg1 NH4-N and 47 mg kg1 available phosphorus.

After being air dried, appropriate concentrations of the mixtures of PHE and PYR (0, 50 + 50,

100 + 100, and 200 + 200 mg kg1) were spiked into soil samples to achieve certain PAH

concentrations. PHE and PYR (>98%) were purchased from Aldrich Chemical Co. Ltd., USA.

The spiked soils were placed in the dark at room temperature for 30 days prior to the

experiments. Before starting the experiment, initial concentrations of PHE and PYR in spiked

soils were tested and recorded as 43.521.34, 71.240.31, and 174.564.14 mg kg1 for PHE,

49.661.36, 93.433.32, and 192.173.27 mg kg1 for PYR in 50 + 50, 100 + 100, and 200 +

200 mg kg1 spiked soils, respectively.

2.2 Microbial inocula and host plants

Rhizobia strain Ensifer sp.4027 (strain CGMCC 10994) was isolated from Sesbania cannabina

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(Sesbania pea) plants collected from Dongying, Shandong province (37.76N, 118.98E). The

original inoculum of the AM fungi Glomus mosseae (BGC NM03D) provided by the Institute of

Plant Nutrition and Resources (Beijing, China) was propagated in pot culture on Trifolium

repens (White clover) for 8 weeks. The sporedensity was 116 spores per 10 ml inoculum. Seeds

of Sesbania cannabina (Retz.) Pers. (Shandong Academy of Agricultural Sciences, Shandong,

China) were surface-sterilized in 10% hydrogen peroxide for 10 min and rinsed several times

with distilled water before use.

2.3 Greenhouse experiment and sampling

Surface sterilized seeds were sown in porcelain pots (15 cm in diameter20 cm high) containing

1.5 kg air-dried soil. After germination, the seedlings were thinned to 3 per pot, followed by

inoculation with 10 ml AMF inoculum and/or 10 ml rhizobia broth (1.2108 CFU ml1) per kg

soil. In the non-inoculation treatments, an equivalent amount of -irradiated-sterilized inocula,

was used instead of the AMF inocula and the rhizobia inoculation was omitted and replaced an

equivalent amount of sterilized broth to provide similar conditions. All the pots were arranged

randomly in a greenhouse, with natural light and day/night temperatures of 30/24C, 60% 2%

relative humidity. AM inoculums and rhizobia were applied as the seeds were sown. Quarter of

the Hoagland solution was supplied regularly and the pots were weighed every week to adjust

the water content.

A total of six treatments with three replicates for each treatment were set up: (1) unplanted soil

(CK); (2) unplanted soil inoculated with rhizobia (R); (3) soil planted with Sesbania cannabina

(S); (4) planted soil inoculated with rhizobia (SR); (5) planted soil inoculated with AMF (SA);

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and (6) planted soil inoculated with rhizobia and AMF (SRA). PAHs-free control was set up for

four planted soil treatments: (3) (4) (5) (6). Additionally, an abiotic control pot containing 10 g

kg1 of sodium azide (microbial inhibitor) was also included in the experiment.

After 60 days of growth, shoots and roots of S. cannabina plants were harvested and washed

with sterile water. Excised nodules and nodulated roots were collected for measurements of

leghemoglobin content and nitrogenase activity respectively. Small amounts of fine roots (< 0.2

g fresh weight) were randomly collected from each pot to determine the mycorrhiza infection

rate. Roots and shoots were freeze-dried and weighed, then ground for PAHs analysis. Soil in

each pot was ground sufficiently to pass through 100-mesh sieve, and divided into two portions.

One was stored at-20C for DNA extraction, and the other portion was stored at 4C for PAHs

and other biogeochemical analysis.

2.4 Leghemoglobin Content of Nodules and Nitrogenase Activity

Measurement of leghemoglobin content was based upon the conversion of hematin to pyridine

hemochromogen, and nitrogenase activity was based on acetylene reduction assay (Ren et al.

2016).

2.5 PAH analysis

5 g of freeze-dried soil sample or 2 g plant samples was mixed with 15 ml dichloromethane:

acetone (1:1) in a glass centrifuge tube and extracted for 20 min with an Ultrasonic Disrupter,

then centrifugation at 3000 rpm for 10 min to precipitate the soil or debris. The supernatant was

collected and concentrated into about 2 ml in a rotary evaporator, dissolved in 10 ml n-hexane

and loaded on to a column packed with layers of silica gel (200-300 mesh), neutral aluminum

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oxide (100-200 mesh) and Na2SO4 followed by elution with 80 ml hexane and dichloromethane

(7:3, v/v) mixture. The filtrate was re-concentrated to 2 ml and further carefully blown dry with

nitrogen. The residue was dissolved in 100 l of n-hexane and filtrated with 0.45 m-Teflon

filter to remove particles prior to analysis.

PAHs were analyzed using an Agilent 7890A gas chromatography equipped with a flame

ionization detector base on Lee et al. (2008). A DB-5 ms (30 m0.25 mm 0.25 m) column

(Agilent, Santa Clara, CA, USA) was used with the following temperature program: column held

at 60C for 1 min after injection, increased by 15C/min to 200C, held for 1 min, increased by

2C/min to 214C, then increased by 5C/min to 290C, and held for 1 min. The retention times

of PHE and PYR were recorded as11.97 and 17.10 min, respectively.

2.6 Microbial numbers and mycorrhizal colonization

To enumerate the PAH-degrading microbe population, aqueous extracts of 1 g soil samples were

serially diluted and spread on basal mineral medium agar plates (NH4Cl, K2HPO4, KH2PO4,

MgSO4, pH 7.2) containing 100 mg L1 PHE, and 50 mg L1 of cycloheximide for suppression of

fungal growth. There are three replicates for each dilution. Plates were incubated about 2 weeks

at 28Cprior to counting the numbers of colony forming units (CFU).

The percentage of mycorrhizal colonization in the roots was calculated by the gridline

intersection method (Giovannetti and Mosse 1980), after staining with trypan blue.

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2.7 Water-soluble phenols content

Water-soluble phenols in soils were quantified colorimetrically according to Carter (Carter

1993). Water-soluble phenols were extracted by 15 ml distilled water; shake for 4 h, followed by

centrifugation at 3000 g for 20 min. A 10 ml aliquot of extract or standard was placed in test

tube, then 3 ml of Na2CO3 solution was added followed by 1 ml Folin-Ciocalteau reagent

(Sigma-Aldrich, USA). Absorbance was recorded at 750 nm. Vanillic acid was used as the

standard, and the amount of phenolic compounds is represented as vanillic acid equivalents (g

vanillic acid g1 soil).

2.8 Enzymatic activity

Soil dehydrogenase activity was measured by the reduction of triphenyl tetrazolium chloride

(TTC) to triphenyl formazan (TPF). 3 g soil sample was incubated for 12 h at 37C in 5 ml of

TTC solution (4 g L1, 0.2 M Tris-HCl pH 7.4). 5ml of toluene added into the sample to extract

TPF, followed by centrifugation at 5000 g for 5 min, and absorbance of color in the extract was

taken at 492 nm. Soil dehydrogenase activity was measured as g TPF g1 dry soil.

Soil polyphenol oxidase (PPO) activity was determined by the colorimetric method of Yu et al.

(2011). The mixture of 1.0 g soil and 10 mL 1% pyrogallic acid was incubated at 30Cfor 2 h,

and 4 mL citric--phosphoric acid buffer (pH 4.5) was added to the mixture. The purpurigallin

(PPG) formed was extracted with chloroform, measured by a spectrophotometer at 430 nm. The

results are expressed as g PPG g1 dry soil.

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2.9 DNA extraction and qPCR assay

In this study, nidA gene is used to quantify pyrene-degrading bacteria in soils during the

biodegradation process (Debruyn et al. 2007). Soil DNA was extracted and purified using a soil

DNA isolation kit (MO-BIO, USA). Real-time qPCR was performed using the DNA Engine

Opticon 2 Real-time PCR Detection System (Bio-Rad, Hercules, CA, USA) and SYBR green

probe (SYBR Premix Ex Taq system, Takara Bio Inc., Otsu, Japan). Specific primers for nidA

were NidA-F: 5-TTCCCGAGTACGAGGGATAC, and nidA-R: 5-

TCACGTTGATGAACGACAAA. PCR amplification conditions were: 94C for 30 s, 95C for

15 s, 56Cfor 1 min. Calibration of qPCR was carried out with a serial dilution prepared with the

appropriate cloned target sequence.

2.10 Statistical analysis

All values from the chemical and biological analyses of soil are represented as means SDs of

three replicates for each treatment. One-way ANOVA and Duncans multiple range test were

used to identify significant differences, using SPSS ver. 16.0 (SPSS Inc., Chicago, IL, USA).

3 Results and discussion

3.1 Mycorrhizal colonization and Nodulation

Mycorrhizal colonization and nodulation were measured after 60 days. The different levels of

PAHs tested did not affect the colonization ability of G. mosseae (GM) significantly. There was

no difference in the mycorrhizal infection levels of S. cannabina roots under the different

conditions of PHE+PYR concentrations used (Table 1). Moreover, the colonization rate was

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elevated when the mycorrhizal plants were inoculated with Ensifer sp.4027 (4027) in PAHs

contaminated soil (Table 1).

To study the effect of AM fungi on rhizobia in dual-inoculated S. cannabina, nodulation and

nitrogen fixation were investigated. Data presented in Table 1 revealed that the number and mass

of nodules significantly decreased in 100+100 and 200+200 mg/Kg PAHs soil in

rhizobia-inoculated and dual-inoculated plants. Interestingly, dual inoculation of 4027+GM

increased the average number of nodules compared with the 4027 plants under same PHAs

concentration. Total nodule weight per plant followed a similar pattern to the number of nodules.

The leghemoglobin content in rhizobia-inoculated plants was sharply reduced in PAHs

contaminated soil, however, significantly less damage to the leghemoglobin protein was

recorded in 4027+GM-inoculated plants compared with 4027-inoculated plants. Nodule activity,

according to the ethylene transformation from acetylene, corresponded to the reduction in nodule

dry mass and leghemoglobin content in PAHs spiked soils (Table 1).

It is well known that the plant--rhizobium system benefits from the presence of AM fungi

because the mycorrhizae ameliorate not only P deficiency but also any other nutrient deficiencies

that might be limiting to rhizobial symbiont (Xiao et al. 2010). Nautiyal et al. (2010) found that

the dual inoculation of Cicer arietinum L. with rhizobia and AMF significantly enhanced the

number of nodules and the dry weight per plant. Lisette et al. (2003) reported that co-inoculation

with rhizobia and compatible AM fungi could dramatically enhance pea growth, and N and P

uptake. However, previous works on the tripartite symbiosis legume--mycorrhiza--rhizobia have

shown not only stimulatory effects (Xiao et al. 2010), but also inhibitory effects on each other or

on the growth of plants (Franzini et al. 2010). Studies have shown that some bacterial species

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respond to the presence of certain AM fungi (Artursson et al. 2006), suggesting a high degree of

specificity between bacteria associated with AM fungi. The present study showed that 4027 and

GM were mutually promoted, therefore, had great potential to improve S. cannabinas growth in

PAHs spiked soil.

3.2 Plant biomass

Plant biomass was measured to explore the tolerance of S. cannabina growing in a PAHs

contaminated soil. The dry weight of shoot and root biomasses of plants grown in the PHE and

PYR spiked soils are shown in Table 2. The different treatments tested had distinct outcomes.

Root and shoot yields of all S. cannabina were significantly lower in 100+100 or 200+200

mg/Kg PAHs compared with that in PAHs free soil. Inoculation of 4027 and/or GM significantly

increased root and shoot biomass yields of S. cannabina (Table 2). Results also revealed that

root/shoot ratios of S. cannabina plants decreased as a result of increase in PAHs amendment of

the soil. The decrease in the root/shoot ratios of S. cannabina plants can be attributed to the

apparent more negative effects of PAHs on the roots compared to the shoot.

The reduction in S. cannabina plants biomass might result from the inherent toxicity of PAHs.

Plants are sensitive to low-molecular-weight volatile hydrocarbons, which are soluble and can

easily diffuse though the plasma membrane and get inside plant tissues. Reilley et al. (2011)

suggested that PAHs might reduce the water and nutrients available for plants in polluted soil,

leading to a decline in plant yield. Although biomass was reduced in PHE and PYR spiked soil,

the plants investigated did not exhibit visible signs of stress; therefore, it is feasible to use S.

cannabina for vegetation establishment in PAHs-contaminated soils.

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3.3 PAH accumulation in plants

PHE and PYR concentrations were detected in S. cannabina plants grown in the spiked soils, the

results are shown in Figure 1. In all planted treatments, PAHs accumulation in roots was higher

than in shoots. Other have previously found that PAH translocation from roots to shoots was

minimal (Yu et al. 2011). In the present study, inoculation with 4027 and GM alone or together

significantly enhanced PAHs accumulation inside roots (Figure. 1), but not shoots. Several other

studies have also shown that the accumulation of PAH in root tissues was significantly greater in

mycorrhizal compared to non-mycorrhizal plants (Yu et al. 2011). This enhancement can be

attributed to the increased biomass resulting from the presence of mycorrhiza or from nodule

formation; or the chemical composition change of plant roots due to AMF inoculation and the

increased root access to soil due to the formation of the fine soil pores interwoven by AM

hyphae (Teng et al. 2011).

Although substantial amounts of PHE and PYR were accumulated in the roots, the percent

removal of PHE and PYR by the S. cannabina plants was only 0.016--0.079% for the four

planted treatments (Figure 2). These results illustrate the minimal contribution of phytoextraction

to soil PAHs depletion compared to biodegradation process in soils, which was also previously

noted by Teng et al. (2011).

3.4 Soil PAH removal

Residual concentrations of PHE and PYR remaining in soil at the end of experiment are shown

in Figure 3. After 60 days of incubation, the PAHs dissipation rates were 81-99% for PHE,

72-85% for PYR in four planted treatments (S, SR, SA and SRA). The self-dissipation rates of

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untreated CK were 59-63% for PHE and 49-54% for PYR, probably due to the activity of

indigenous microbial degraders. In the abiotic control soil which was mixed with sodium azide

(10 g kg1dry soil), only 5.2% of the total PAHs were removed after 60 days experiment though

abiotic pathways (data not shown). Furthermore, there were significant differences in PAHs

removal between planted and unplanted soils. The highest PAHs dissipation rate was observed in

the planted soil inoculated with 4027 and GM. The percentage removal of PAHs increased

significantly in the planted treatment receiving AMF (SA), relative to the planted treatment

without AMF (S). The dual-inoculation of 4027 and GM further enhanced PAHs removal in the

SA group. These results indicate that the combined application of rhizobia and AMF had a

greater stimulatory effect on PAHs removal than the single inoculation of either of them alone.

Notably, rhizobia 4027 treated soil (R) had higher PAHs dissipation rates than the untreated

control (CK).

The use of mycorrhizae to improve phytoremediation of organic pollutants has recently received

great attention. Yu et al. (2011) found that arbuscular mycorrhizal ryegrass significantly

enhanced the dissipation of pyrene from soils. Aranda et al. (2013) observed increased PAHs

dissipation in the mycorrhizal roots culture medium and the dissipation rate was affected by the

PAHs concentration and type.

Rhizobium, particularly the naturally symbiotic types, had great potential to enhance the

phytoremediation of polluted soils (Johnson et al. 2005; Xu et al. 2010). Legume--rhizobium

symbiosis may be crucial for plant establishment on contaminated sites (Carrasco et al. 2005).

Several studies have indicated that rhizobia can promote the production of secondary metabolites

in the host plant roots exudates, such as flavones, which control nodABC expression during

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nodule development (Peters et al. 1986). Thus, increased roots exudation may in turn stimulate

the growth of microbial degraders or influence pollutant bio-availability. Johnson et al. (2005)

observed that R. leguminosarum bv. trifolii could enhance plant vigour and growth when

inoculated to its host plant, and played an important role in the rhizoremediation of recalcitrant

PAHs. It follows that increasing of plant root exudates by inoculation with rhizobia may in turn

lead to the promotion of the growth of PAHs-degraders. Previous studies also have indicated that

rhizobia, either as free living cells or in symbiosis with plants, have the ability to transform PCBs

and PAHs (Yu et al. 2011; Aranda et al. 2013). In the present study, rhizobia 4027 treated soil

(R) had higher PAHs removal than untreated control (CK).

3.5 Water-soluble phenolic compounds

Water-soluble phenolic compounds (WSP) are an important class of compounds exuded by plant

roots. WSP concentrations in soils were monitored to evaluate the root exudation of S. cannabina

response to PHE and PYR addition. Throughout the 60 days growth period, the WSP

concentrations of PAHs soil were significantly higher in all four planted treatments, compared to

the PAHs-free soil (Table 3). The results are in line with previous finding that soil WSP

concentrations positively correlate with PHE and PYR in planted soils (Lee et al. 2008). In

addtion, inoculations of 4027 and GM significantly increased WSP accumulation in PAHs soil,

but had no impact in PAHs-free soils (Table 3). The majority of phenolic compounds were

products of the shikimate and acetate pathways in plants with synthesis/accumulation tended to

be enhanced by environmental stress (Siqueira et al. 1991). Rhizobia and AMF may help S.

cannabina to strengthen this physiological response in order to overcome persistent aromatic

pollutants. Rhizosphere microorganisms able to catabolize phenolic compounds as carbon source

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often possess enzymes that can co-metabolize pollutants with similar aromatic ring structures

(Chaudhry et al. 2005). Phenolic compounds released into the soil might result in selective

growth and long-term survival of certain soil microbes, a situation conducive to the enhancement

of rhizosphere-facilitated degradation of recalcitrant pollutants such as PAHs.

3.6 Microbial enumeration and qPCR assay of nidA

Soil PAH-degrading bacterial counts in PAHs spiked soils of different treatments are presented

in Figure 4A. The soil used in this study contained a large amount of indigenous PAH degraders

(1.07106 CFU g1 soil). This likely ensured the success of soil phytoremediation without

inoculation of exogenous degrading bacteria. Statistically significant differences in the

concentration of degraders (p < 0.05) were observed 15 days after incubation between unplanted

(CK) and planted (S, SR, SA and SRA) treatments (Figure 4A). In the CK group, the degraders

increased to the highest density at day 30, and then decreased gradually to eventually stabilize at

106 CFU g1 dry soils. In contrast, planted treatments maintained higher population of degraders

(107-109 CFU g1 dry soil) until harvest. Root exudation creates quantitative and qualitative

spatial gradients that could positively affect microbial counts and activities in soil (Lu et al.

2010). The decrease in the concentration of degraders detected at the later stages of our

experiments could result from nutrient shortage due to the depletion of available nutrients in soils

(Lu and Lu 2015). As expected, according to the statistical results, rhizobia and AMF inoculation

significantly (p < 0.05) increased the bacterial population than S. cannabina along (Figure 4A).

In addition to the promotion on S. cannabinas growth and root secretion by rhizobia and AMF,

the exudates from AMF extracellular hyphae can be used as substrates by soil bacteria. Toljander

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et al. (2007) found that AMF exudates increased bacterial growth and altered bacterial

community composition in soil.

It is known that nidA gene encodes the large subunits of pyrene dioxygenase in some

Mycobacterium species (Debruyn et al. 2007). The nidA dioxygenase is responsible for both the

initial dioxygenase step of pyrene and the phenanthrene degradation pathway (Stingley et al.

2004). In this study, nidA gene was quantified using real-time qPCR method to evaluate changes

in the PAHs degraders. The nidA gene copy numbers during the 60 days of incubation is shown

in Figure 4B. The numbers of nidA gene copy of unplanted soil increased gradually during the

experiment. The detection of nidA gene in the soil indicates the existence of potentially the

PAH-degrading Mycobacterium. S. cannabina cultivation, rhizobia and AMF inoculation

obviously accelerated the proliferation of the nidA population.

3.7 Soil enzyme activity

Soil enzyme activity in PAHs spiked soil was monitored during the phytoremediation process to

determine how PAHs and plants affect microbial activity. The dehydrogenase activities in

planted and unplanted soils are shown in Figure 5. During the 60 days incubation, the

dehydrogenase activity in planted soils was significantly higher compared to unplanted soils (p <

0.05), indicating that the activities of the microbial communities in the planted soils were

enhanced by the presence of S. cannabina roots. In the present study, the stimulation of

dehydrogenase activity correlated well with the increased in microbial concentrations (Figure

4A) and elevated WSP (Table 3) in planted soils. Dehydrogenase activity assays in soil have

often been used to obtain correlative information on the biological activity of microbial

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populations in soil, i.e., as an index of total microbial activity. Strong correlations between

hydrocarbon removal and dehydrogenase activity are frequently observed (Margesin et al. 2000).

Soil polyphenol oxidase (PPO) is an oxygen-transferring enzyme which participated in PAHs

degradation in soil (Durn et al. 2000). Our analysis of PPO activities in planted soil (S, SR, SA

and SRA) versus non planted siols revealed that PPO activities were much higher (p < 0.05) in

the former compare to the latter (CK) (Figure 5A). Inoculation with 4027 and GM significantly

increased PPO activity compare with S. cannabina cultivation along. In addition, PPO activity

tended to decrease gradually over time. Similar patterns were observed in other studies (Jing et

al. 2013). Overall, we observed here that changes in PPO activity correlated with changes in the

concentration of the PAH degraders population.

The results above suggest that the tripartite interactions between rhizobia, AMF and S.

cannabina enhanced PAHs degradation via stimulating both microbial development and soil

enzyme activities.

4 Conclusions

This study was conducted to investigate soil PAHs remediation by plants in order to contribute to

the technology and practice of field phytoremediation. S. cannabina was selected because of its

extensive, fibrous root system providing a large root surface area for the growth of microbial

populations. Our results indicate that S. cannabina may play an important role in the depletion of

PAHs in soil. Furthermore, the association between S. cannabina, rhizobia and AMF

significantly increase the concentration of PAHs-degrading bacteria and related microbial

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activities in soils, is thus a suitable alternative for phytoremediation of aromatic compounds

polluted soil.

Acknowledgments:

This work was financed by the Key Research Program of the Chinese Academy of

Sciences (Grant NO. KZZD-EW-14), the National Natural Science Foundation of China

(31370108 and 31570063), One Hundred-Talent Plan of Chinese Academy of Sciences

(CAS), Yantai Science and Technology Project (2013JH021). We thank Professors Hui

Lin and Bing Zhao for kindly providing arbuscular mycorrhiza strains. We thank

Professors Gladys Alexandre and Entao Wang for insightful comments on the

manuscript.

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Table 1 Nodulation, nitrogenase activity, leghemoglobin content and mycorrhizal colonization

of Sesbania cannabina plants inoculated with rhizobium and/or Glomus mosseae grown under

different levels of PHE and PYR. Means with different letters within each column are

significantly different (one-way ANOVA, Duncans multiple range test, P < 0.05). ---

represented no data was collected. SR: planted soil inoculated with rhizobia; SA: planted soil

inoculated with AMF; SRA: planted soil inoculated with rhizobia and AMF.

Inoculatio PHE Nodule/plant Nitrogenas Leghaemoglobi Mycorrhiza

n and e activity n content l
PYR colonizatio
n

(mg Number Fresh (mol C2H4 (mg g1 FW (%)

kg1) weight (g) g1 FW nodules)
nodules
h1)

SR 0+0 37.332.08c 1.030.04 1.820.05e 1.130.05e ---

d e

50+50 39.672.52d 1.010.04 1.880.11e 0.710.09c ---

e

100+10 27.673.53b 0.740.06 1.250.06c 0.520.04b ---

0 c

200+20 182.58a 0.410.05 0.470.03a 0.230.03a ---

0 a

SA 0+0 --- --- --- --- 162a

50+50 --- --- --- --- 133a

100+10 --- --- --- --- 152a

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200+20 --- --- --- --- 114a

0

SRA 0+0 47.332.08e 1.230.07f 2.120.14f 1.130.06e 291d

50+50 52.673.45f 1.320.06f 2.180.15f 0.910.08d 252c

100+10 34.671.47c 0.890.07 1.550.09d 0.720.03c 211b

0 d

200+20 242.36b 0.510.03 0.670.07b 0.430.03b 212b

0 b

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Table 2 Biomass (g dry weight pot-1) of shoots and roots and root shoot ratio (Wroot/Wshoot) of

different treatments in PHE and PYR spiked soils. Uppercase letters indicate significant

differences within each row; different lowercase letters among different treatment of shoot or

root within each column are significantly different (one-way ANOVA, Duncans multiple range

test, P < 0.05).S: soil planted with Sesbania cannabina; SR: planted soil inoculated with

rhizobia; SA: planted soil inoculated with AMF; SRA: planted soil inoculated with rhizobia and

AMF.

Treatments PHE+PYR(mg Kg1)

0+0 50+50 100+100 200+200

S Shoot 5.270.21Ba 5.420.43Ba 4.930.17ABa 4.40.33Aa

Root 6.20.16Ca 6.310.23Ca 4.660.34Ba 4.060.31Aa

Wroot/Wshoot 1.18 1.16 0.95 0.92

SR Shoot 6.140.23Bb 6.360.28Bb 5.300.22Ab 4.970.23Ab

Root 6.80.21Cb 6.740.15Cb 5.530.41Bb 4.850.26Ab

Wroot/Wshoot 1.11 1.06 1.04 0.98

SA Shoot 6.860.37Cc 6.550.13Bc 6.370.1Bc 5.850.19Ac

Root 8.390.32Dc 7.420.28Cc 6.810.22Bc 5.940.41Ac

Wroot/Wshoot 1.22 1.13 1.07 1.02

SRA Shoot 8.320.15Bd 8.370.35Bd 8.160.4ABd 7.820.42Ad

Root 9.220.17Cd 9.110.37Cd 8.160.14Bd 7.270.35Ad

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Wroot/Wshoot 1.11 1.09 1.00 0.93

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Table 3 Changes of the water-soluble concentration of phenolic compounds (g vanillic acid

g1 soil) in soils with/without PAHs. PAH+, 200mg/Kg PHE and 200mg/Kg PHE spiked soil;

PAH-, PAHs free soil. Uppercase letters indicate significant differences among treatments within

columns; lowercase letters indicate significant differences between PAH+ and PAH within

rows at each sampling time (one-way ANOVA, Duncans multiple range test, P < 0.05).

Treatm 15 days 30 days 45 days 60days

ents

PAHs- PAHs+ PAHs- PAHs+ PAHs- PAHs+ PAHs- PAHs+

S 1.220. 1.590.1 1.730.1 2.260. 1.920.1 2.720. 2.330. 3.570.

1Aa 3Ab 3BCa 12Ab 9ABa 12Ab 12Aa 06Ab

SR 1.180. 2.540.1 1.430.1 2.930. 1.690.1 3.470. 2.430. 3.940.

02Aa 5BCb Aa 13Bb 7Aa 12Bb 11Aa 09Bb

SA 1.20.0 2.810.1 1.590.1 3.040. 2.040.1 4.360. 3.760. 4.970.

6Aa 8Cb 4ABa 12Bb BCa 14Cb 18Ba 06Cb

SRA 1.270. 2.240.2 1.870.1 2.860. 2.320.1 4.510. 3.120. 5.320.

11Aa Bb 9Ca 12Bb 2Ca 13Cb 13Ca 13Db

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Figure 1 Dissipation rates of PHE and PYR from spiked soil. Bars with different letters among

treatments of same PAH level are significantly different (one-way ANOVA, Duncans multiple

range test, P < 0.05). CK: unplanted control soil; R: unplanted soil inoculated with rhizobia; S:

soil planted with Sesbania cannabina; SR: planted soil inoculated with rhizobia; SA: planted soil

inoculated with AMF; SRA: planted soil inoculated with rhizobia and AMF.

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Figure 2 Concentrations of PAHs in plant tissue under different treatments after 60 days. Bars

with different letters among treatments of same PAH level are significantly different (one-way

ANOVA, Duncans multiple range test, P < 0.05). S: soil planted with Sesbania cannabina; SR:

planted soil inoculated with rhizobia; SA: planted soil inoculated with AMF; SRA: planted soil

inoculated with rhizobia and AMF.

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Figure 3 The percent phytoextraction of PAHs from spiked soils under different treatments after

60 days. Percentage of PAHs phytoextracted is the ratio of PHE+PYR content in plant tissues to

total dissipation amount from soil. Bars with different letters among treatments of same PAHs

level are significantly different (one-way ANOVA, Duncans multiple range test, P < 0.05). S:

soil planted with Sesbania cannabina; SR: planted soil inoculated with rhizobia; SA: planted soil

inoculated with AMF; SRA: planted soil inoculated with rhizobia and AMF.

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Figure 4 Changes of nidA gene copies and PAHs-degrading bacteria count in 200 soils during

the 60 days incubation. A: Count of soil PAH-degrading bacteria, B: total nidA gene copies.

Data are meansstandard deviations. CK: unplanted soil; S: soil planted with Sesbania

cannabina; SR: planted soil inoculated with rhizobia; SA: planted soil inoculated with AMF;

SRA: planted soil inoculated with rhizobia and AMF. PAHs concentrations are 200mg/Kg PHE

and 200mg/Kg PHE.

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Figure 5 Changes of soil dehydrogenase and polyphenol oxidase activity during the 60 days

incubation. A: activity of dehydrogenase, B: activity of polyphenol oxidase. Data are means

standard deviations. CK: unplanted soil; S: soil planted with Sesbania cannabina; SR: planted

soil inoculated with rhizobia; SA: planted soil inoculated with AMF; SRA: planted soil

inoculated with rhizobia and AMF. PAHs concentrations are 200mg/Kg PHE and 200mg/Kg

PHE.

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