INTRODUCTION

Modern agricultural practices and increased anthropogenic activities have resulted in high levels of metal pollutants that have significant environmental impacts,consequently affecting human health via food chain(Yu et al.,2014).These metals also have detrimental effects on the soil microbial community,thus reducing ecosystem functioning(Kossoffet al.,2014).Lead(Pb)is one such heavy metal,which has received extensive attention because of its acute and chronic toxicological effects on plants and animals(Cheng et al.,2015).Cleaning up soils contaminated with heavy metals by traditional or conventional techniques,such as chemical precipitation, filtration,ionexchange,reverse osmosis,membrane technology,and electrochemical treatment,can be exorbitant and destructive to the soil(Ahluwalia and Goyal,2007).Remediation of contaminated soils using earthworms and plants appears to be a cost-effective and eco-friendly technology.Most metal-accumulating plants are characterized by slow growth and low biomass yield and,thus,are not true hyperaccumulators capable of accumulating high concentrations of phytotoxic metals in them(Kom´arek et al.,2007).A plant used for phytoextraction should be able to grow rapidly,produce large biomass,and accumulate high concentrations of metals.Pennisetum purpureum is a monocot perennial grass belonging to the family Poaceae(Khan et al.,2007).This species exhibits high biomass production,has low water and nutrient requirements(Strezov et al.,2008),and therefore was selected for the present study to bioremediate contaminated soil.

A few studies have evaluated the combined effects of earthworms and plants on the uptake of heavy metals(Morgan and Morgan,1988;Brokbartold et al.,2012).Earthworms improve soil structure(Lemtiri et al.,2014).They are considered a suitable organism for biomonitoring the effects of heavy metals in contaminated soils(Peijnenburg and Vijver,2009).Recently,bioaugmentation of microbes has gained attention in the bioremediation of toxic pollutants.To intensify the clean-up process,microbes can be coupled with the living biomass systems(Glick,2003).Because microbes have an intimate association with host plants and earthworms,they could be used as reliable bioinoculants to improve the bioremoval of heavy metals.There are a few microbes,including bacteria,which possess a variety of protective mechanisms against very high levels of Pb without having any effect on their growth and metabolism.These unique characteristics of microbes make them an ideal tool for the bioremediation of Pb(Naik et al.,2012).

To the best of our knowledge,the present study was conducted to address the earthworm-facilitated metal extraction by plants bioaugmented with bacteria for the first time.Pennisetum purpureum has been devised as a hyperaccumulator plant concomitant with earthworm(Lumbricus terrestris)bioaugmented with Pb-resistant bacteria(LRB)for the bioremoval of Pb.In order to gain a better understanding of metal uptake by plants and earthworms in contaminated soils in the presence of bacteria,the aims of the present study were to∶i)evaluate the effects of Pb concentrations on physical parameters of plants,ii)assess the toxic effects of Pb on survival,body weight,locomotion,and release of malondialdehyde(MDA)in earthworms under Pb-stressed condition,and iii)investigate the total Pb uptake by plants and earthworms in the presence of LRB.

MATERIALS AND METHODS

Plant,earthworm,and soil sampling

Fresh plant samples were collected from a farm near Brahmapuram,Tamil Nadu(TN),India(12.9657°N,79.1676°E)and were pruned to a uniform root and shoot length of 5 cm for pot culture study.The plants were washed with tap water and rinsed with de-ionized water to remove dirt,soil particles,and debris that might have been deposited on the leaves from the soil and atmosphere in order to avoid the addition of nonindigenous microorganisms to the system.The species L.terrestris,about 6-months-old,was obtained from a vermiculture plant,allowed to grow and adapt to the soil condition provided in the lab for up to 20 d,and used in the present study.The earthworms were kept alive in moist soil in the dark in the laboratory(Richardson et al.,2015).Environmental conditions were maintained at 18°C and 80%humidity.Soil moisture was maintained at 20%soil weight.

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Contaminated soil samples were collected from nearby industrial areas in Erode District,TN,India(11.5246°N,77.4702°E)with a history of heavy metal contamination by a randomized sampling procedure.The samples were collected from a depth of 10 cm from the surface at an equidistance of 1 m in each square of 10 m and packed in sterile polyethylene bags.The soil was passed through a 6-mm mesh and homogenized.The samples were processed immediately after collection(Pouyat et al.,2015).Complete pro filing of the contaminated soil was performed by the Fertiliser Development and Consultation Organization(FDCO)at National Agro foundation,Chennai of India.The physicochemical properties of soil,such as pH,electrical conductivity,and total organic C,total N,and Pb contents,were analyzed(Table I).

TABLE I Physicochemical properties of the contaminated and autoclaved soils used in pot-culture study

a)EC=electrical conductivity;OM=organic matter;Exc.=exchangeable;Ava.=available;CEC=cation exchange capacity.

Propertya) Unit Soil Contaminated Autoclaved pH 7.56 6.11 EC mS cm−1 3.14 0.50 OM g kg−1 70.4 12.1 NO3-N mg kg−1 32.1 52.6 Ava.P mg kg−1 22.04 29.77 Exc.K mg kg−1 133 101 Exc.Ca mg kg−1 2035 835 Exc.Mg mg kg−1 271 242 Ava.S mg kg−1 444.5 35.9 Exc.Na mg kg−1 1561 160 CEC cmol kg−1 19.56 7.13 Ava.Pb mg kg−1 68.96 0.22 Ava.Cu mg kg−1 8.36 2.83 Ava.Zn mg kg−1 4.39 3.97 Ava.Mn mg kg−1 54.23 24.26 Ava.Fe mg kg−1 71.26 20.24

Isolation and screening of the effective LRB isolates from soil

A reduction in number of colonies was observed on plates isolated from the gut of earthworms at Pb of 150 mg kg−1dw compared with Pb of 50 mg kg−1 dw.Moreover,with LRB-augmentation,there was a very high number of bacterial colonies.However,a favorable number of colonies were observed for the control without Pb(Table III).

Isolation of genomic DNA from the isolate and amplification of Pb-resistant gene

Cells were harvested from 10 mL of overnight cultures,and the pellets were lysed in lysis buffer containing 25%sucrose,25 mmol L−1ethylenediaminetetraacetic acid(EDTA),50 mmol L−1Tris-HCl,and 5 mg L−1lysozyme.The chromosomal DNA was extracted by the phenol-chloroform method(Oh et al.,2011).The Pb-resistant gene,pbrA,was amplified using the primers pbrA1(5′-ATGAGCGAATGTGGCTCGAAG-3′)and pbrA2(5′-TCATCGACGCAACAGCCTCAA-3′)(Borremans et al.,2011).The reaction conditions for amplification were as follows∶predenaturalization at 95°C for 4 min,35 cycles of denaturing at 95 °C for 30 s,annealing at 60 °C for 1.5 min,extension at 72°C for 1.5 min,and a final step for extension at 72°C for 7 min.

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Biochemical,morphological,and molecular characterization of the LRB

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Experimental setup

A pot experiment was conducted using soil collected at a depth of 10 cm from the nursery in Vellore Institute of Technology,Vellore,India.A total of 10 kg dry weight(dw)soil was transferred into 10 L rectangular plastic pots,and 10 plantlets were propagated in each pot.After adaptation for 3 d,the soil was artificially contaminated with a solution of Pb(NO3)2to a concentration of 50,100,and 150 mg kg−1dw,respectively,with one pot maintained as the control without Pb.The pots were divided into two sets.In one set,the plant was augmented with 10 earthworms and supplemented with LRB(P+E+B).The other set was also augmented with 10 earthworms but without any bacterial augmentation(P+E).Bioaugmentation was implemented every 10 d in order to maintain an elevated microbial count throughout the experiment.Lead-resistant bacteria were added to the pots as 10 mL of cell suspension(4.0 × 1011–1.0× 1012colonyforming units(CFU)mL−1).All the pots received the same amount of sterile distilled water.Plants grown on Pb-contaminated soil were harvested on Days 0,30,and 60 of growth.Each treatment was carried out in triplicates.The complete physicochemical analysis of the soil used in the experiment before being artificially contaminated by Pb was also carried out by FDCO.The results of the physicochemical analysis of experimental soil are presented in Table I.

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Plant analyses

Morphological parameters and total chlorophyll content. On Day 30,one plant per set,including the control,was uprooted,and its root and shoot lengths were recorded(in cm).The shoot sample was thoroughly washed with distilled water and homogenized with acetone for assessing the total chlorophyll content from chlorophyll a(Chl a)and chlorophyll b(Chl b).The concentrations of Chl a and Chl b(g L−1)were determined using an ultraviolet spectrophotometer at an absorbance of 663(A663)and 645 nm(A645),respectively(Bettini et al.,2016)∶

Measurement of MDA contents. Total MDA content was estimated by measuring lipid peroxidation(Li et al.,2015).Macerated gut tissue(0.1 g)of earthworms was homogenized using 2 mL of 5%(weight∶volume)trichloroacetic acid in an ice bath and centrifuged at 10000× g for 10 min at 4°C.About 2 mL of the supernatant was mixed with 2 mL of 0.67%(weight∶volume)thiobarbituric acid.The mixture was incubated in a boiling water bath for 30 min,cooled,and centrifuged.The absorption of the supernatant was carried out,and the abserbance at 450,532,and 600 nm was used to calculate MDA content(µmol g−1)(Li et al.,2015).

The LRB culture supplemented was biochemically tested by IMVIC(indole,methyl red,Voges-Proskauer,and citrate),catalase,and oxidase tests.Morphological characterization was performed by motility,capsule,and Gram staining as per the standard methods(Cappuccino and Sherman,1992).The bacterial isolate was identified using 16S rRNA gene sequencing using at a commercial facility of Chromous Biotech,India using the universal primers 27F(5′-AGAGTTTGATCCTGGCTCAG-3′)and 1492R(5′-GGTTACCTTGTTACGACTT-3′).The sequences were compared using the basic local alignment search tool(BLAST),and related sequences were obtained from the sequence database for comparison.Multiple alignments were performed,and phylogenetic trees were constructed by the maximum parsimony method using the default parameters of MEGA4 and bootstrapped using 1000 bootstrap trials(Tamura et al.,2007).

Total phenolic and flavonoid contents. The total phenolic content of the extract was determined by the Folin-Ciocalteu method(Kaur and Kapoor,2002).The crude extract was made up to 5 mL(1 mg mL−1)with distilled water and mixed with 0.5 mL of Folin-Ciocalteu reagent,followed by the addition of 2 mL of 20%(weight∶volume)Na2CO3.The mixture was allowed to stand in the dark for 60 min.The absorbance was recorded at 650 nm.The total phenolic content was calculated from a calibration curve,and the results were expressed as mg gallic acid equivalent(GAE)g−1dw.The total flavonoid content of the crude extract was determined by the AlCl3colorimetric method(Chang et al.,2002).Fifty microliters of the extract was made up to 1 mL with methanol,and mixed with 4 mL of distilled water and 0.3 mL of 5%(weight∶volume)NaNO2solution.About 0.3 mL of 10%(weight∶volume)AlCl3solution was added 5 min after incubation,the mixture was allowed to stand for 6 min,and then 1 mol L−1NaOH solution was added.The final volume of the mixture was made up to 10 mL with double distilled water.The mixture was allowed to stand for 20 min,and the absorbance was recorded at 510 nm.The total flavonoid content was calculated from a calibration curve.The result was expressed as mg rutin equivalent(RE)g−1dw.

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Root bio film formation. Bio film formation by the root microbiota was analyzed by the microtiter plate method.Roots of various samples were scraped into individual test tubes containing 5 mL of water.The samples were loaded into each well of a microtiter plate and incubated overnight.The samples were removed,and the wells were carefully rinsed with sterile distilled water and air dried.Crystal violet(1%,weight∶volume)was added to the wells,incubated for 45 min,and washed with distilled water.Subsequently,95%ethanol was added to the wells and incubated for 30 min,followed by transfer of the sample to a new microtiter plate.Optical density reading was taken at 570 nm(Djordjevic et al.,2002).

Earthworm analyses

The earthworms were hand-collected at an interval of 30 d,and its mortality was determined along with other morphological factors.

Physical parameters of earthworms and enumeration of gut microbes. Various physical characteristics,such as length,body weight,and locomotion of the earthworms were recorded before and after the treatment with different concentrations of Pb and microbes.The locomotion was determined based on the distance covered by the earthworms in 1 min(Lemtiri et al.,2016).

It was found that with LRB augmentation,the phenolic and flavonoid contents of the plants showed a differential response over time.The phenolic and flavonoid content was found to be higher in plants not augmented with LRB when compared to the plants augmented with LRB.The highest total phenolic content of the methanolic root extract,calculated from the calibration curve(R2=0.998),was 48.01±1.2 mg GAE g−1dw,and the total flavonoid content(R2=0.999)was 56.01 ± 1.6 mg RE g−1dw at 50 mg kg−1dw Pb in the P+E set(Table II).

For the enumeration of gut microbes,three earthworms from each tray,including the control,were sacrificed 30 d after the treatment.The gut region was separated from the rest of the body parts,and macerated using mortar and pestle with distilled water.Upon serial dilution,10−3dilution was spread plated on the nutrient agar plate and incubated at 30°C for 24 h(Thakuria et al.,2010).

Preparation of plant extract. The roots and shoots of the plant were dried in shade at 25°C and ground to a fine powder in a mechanical blender.The tissue powder was dried,packed into a Soxhlet apparatus,and extracted with 200 mL methanol for 3–4 h at 60°C.The extract was filtered through a Whatman filter paper No.1,and concentrated under reduced pressure at 40°C.The extract was dried,weighed,and stored at 4°C in storage vials further use(Baba and Malik,2015).

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Quantification of acid-extractable Pb

The plant samples(roots and shoots),earthworms,and soil samples from each set on Days 0,30,and 60 were subjected to atomic absorption spectroscopy(AAS)to assess the total Pb uptake.The samples were acid digested following the Environmental Protection Agency(EPA)method 3051A.One gram of plant tissue was used for acid digestion.Earthworm typically weighing between 100 and 400 mg of dw was lyophilized,and used for the analysis.The sample was acid digested with a strong acid(9∶1,70%HNO3∶30%HCl,volume∶volume)(Thermo Fisher Scientific,Cambridge,USA)(Richardson et al.,2015)and processed using Varian’s atomic absorption spectrophotometer at the Technology Business Incubator,Vellore Institute of Technology University(Sardans et al.,2010).Bioaccumulation factor(BAF)was calculated to estimate total Pb bioavailability in the earthworms(Lee et al.,2009)∶

where Mwormis the metal concentration in the earthworm tissue(mg kg−1dw)and Msoilis the metal concentration in the soil(mg kg−1dw).The concentration of Pb in the samples was estimated in triplicate.

Statistical analysis

Statistical analysis was performed using GraphPad InStat software.Significant differences among treatments were analyzed by one-way analysis of variance(ANOVA),using P＜0.05 as a significant level.

RESULTS

Isolation of effective LRB isolates from soil and amplification of Pb-resistant gene

In the present study,the population of bacteria in the sample ranged from 300 to 400 colonies mL−1.A total of 10 bacterial single colonies were isolated,out of which only one isolate,VITMVCJ1,was able to tolerate Pb up to a maximal concentration of 1000 mg L−1.It was considered to be the most effective.The isolate was characterized by pale white,round,and elevated colonies(Fig.1a).Polymerase chain reaction(PCR)-amplified products of VITMVCJ1 yielded the expected 750 bp amplicons,indicating the presence of Pb-resistant gene,and thus was used in the further analyses(Fig.1b).

Biochemical,morphological,and molecular characterization of VITMVCJ1

The isolate VITMVCJ1 was found to be nonmotile,encapsulated,Gram-negative,rod-shaped bacterium showing a positive result for Voges-Proskauer and citrate tests,and a negative result for indole production and methyl red test.Furthermore,VITMVCJ1 showed the highest(99%)nucleotide sequence identity with that of the nearest neighbor Klebsiella variicola,belonging to the family Enterobacteriaceae(Fig.2).The gene sequence has been submitted to Gen-Bank and has been signified with the accession number KU359261.

Plant morphological parameters and total chlorophyll content

Bioaugmentation had a positive effect on plant biomass production.There was an initial increasing trend in plant biomass,which became statistically significant in the shoots and roots after 5 d in the P+E+B set.The shoot and root lengths of the plant was enhanced by 12.5%and 11.6%,respectively,after 60 d in the P+E+B set.In the P+E set,the growth of the plant was meagre with increasing concentration of Pb without bioaugmention.The highest concentration of Pb(150 mg kg−1dw)was virtually lethal for the plants after 30 d,and their survival rate was significantly reduced(Fig.3).There was a significant decrease in the total chlorophyll content of the plants in the P+E set.Interestingly,when bioaugmentation was performed,these parameters were appeared to be favored.There was an upsurge in the chlorophyll content of plants grown in pots with bacterial inoculum(Fig.4).

Plant total phenolic and flavonoid contents

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Root bio film assay

Fig.1 Minimum inhibitory concentration(MIC)of Pb for the bacterial isolates(a)and polymerase chain reaction amplified-products by Pb-resistant gene(pbrA)(b).Lane M=1 kb DNA ladder;Lane 1=Pb-resistant gene amplified in the effective VITMVCJ1 isolate.Vertical bars are the standard deviations of means.

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From the microtiter method of bio film detection,the roots collected from the plants in the P+E+B set were found to be strong bio film producers with higher absorbance at 570 nm when compared with the roots from the plants grown in the P+E set(Fig.5).Earthworm mortality and physiological parameters of the earthworms

Fig.2 Phylogenetic relationship of the isolate VITMVCJ1 with other reported sequences.The bootstrap consensus tree inferred from 1000 replicates was taken to represent the evolutionary history of the taxa analyzed.

Fig.3 Variation in the root and shoot lengths of the plants in experimental pots of two sets(P+E and P+E+B)arti ficially contaminated with different Pb concentrations,measured on Days 0,30,and 60 of growth.Vertical bars are the standard deviations of means.Control=without Pb supplementation;P+E set=plants augmented with earthworms;P+E+B set=plants augmented with earthworms and Pb-resistant bacterium.

Fig.4 Total chlorophyll content in shoots of the plants in experimental pots of two sets(P+E and P+E+B)artificially contaminated with different Pb concentrations,measured on Days 30 and 60 of growth.Vertical bars are the standard deviations of means.Control=without Pb supplementation;P+E set=plants augmented with earthworms;P+E+B set=plants augmented with earthworms and Pb-resistant bacterium.dw=dry weight.

The results of earthworm life-cycle parameters were subjected to ANOVA to verify if there was a change in mortality 60 d after exposure to Pb.None of the earthworms died in the control soils throughout the experimental period,signifying that the experimental conditions were suitable for the survival of the earthworms.However,without LRB augmentation,earth-worms could not survive 30 d of the experiment,compared to a decrease in the mortality rate of earthworms with LRB augmentation.There was a significant difference in the characters between treated(with LRB)and untreated earthworms.Though there was no significant difference in the length of earthworms,the mean body weight of the earthworms was found to be higher with LRB augmentation.Without LRB,there was a significant reduction in body weight and locomotion with increase in the concentration of Pb.Though the reduction was not uniform,it clearly indicated the differential response of the earthworms to the varying concentrations of Pb(Figs.6 and 7).

TABLE II Total phenolic and flavonoid contents in the methanolic root extract of plants in the experimental pots of two sets(P+E and P+E+B)a)artificially contaminated with different Pb concentrations of 50,100,and 150 mg kg−1dry weight(dw)

a)P+E set=plants augmented with earthworms;P+E+B set=plants augmented with earthworms and Pb-resistant bacterium. b)Gallic acid equivalent. c)Rutin equivalent. d)Control=without Pb supplementation. e)Mean±standard deviation(n=3).

Set Total phenolic Total flavonoid content content mg GAEb)g−1dw mg REc)g−1dw Controld) 25.00±1.7e) 38.00±1.2 P+E,50 48.01±1.2 56.01±1.6 P+E,100 52.04±1.7 53.01±1.7 P+E,150 63.17±1.8 60.14±1.8 P+E+B,50 26.05±1.2 32.14±1.3 P+E+B,100 30.16±1.5 38.15±1.6 P+E+B,150 33.18±1.7 40.19±1.8

Fig.5 Root bio film assay of plants in the experimental pots of two sets(P+E and P+E+B)artificially contaminated with different Pb concentrations,expressed as absorbance at 570 nm.Vertical bars are the standard deviations of means.Control=without Pb supplementation;P+E set=plants augmented with earthworms;P+E+B set=plants augmented with earthworms and Pb-resistant bacterium.dw=dry weight.

Fig.6 Variation in the body weight of the earthworms in the experimental pots of two sets(P+E and P+E+B)artificially contaminated with different Pb concentrations,measured on Days 0,30,and 60 of growth.Vertical bars are the standard deviations of means.Control=without Pb supplementation;P+E set=plants augmented with earthworms;P+E+B set=plants augmented with earthworms and Pb-resistant bacterium.dw=dry weight.

Enumeration of microbes in the gut of earthworms

The samples were serially diluted and spread plated on Luria Bertani(LB)medium supplemented with various concentrations of Pb in the form of Pb(NO3)2 ranging from 100–1000 mg L−1(Zhang et al.,2011),and incubated at 30°C for 24 h.Morphologically distinct colonies were purified and maintained in glycerol stock.To obtain the most effective LRB isolates,minimum inhibitory concentration(MIC)was determined(Chatterjee et al.,2012).The pure bacterial cultures were inoculated in the LB broth media with varying concentrations of Pb(250,500,750,and 1000 mg L−1,respectively)and checked for turbidity after 24 h.

Estimation of MDA content in earthworms

The highest level of MDA content was found on Day 30 in earthworms without LRB augmentation,which was 32%higher than that with LRB augmentation.Subsequently,MDA content tended to decrease with exposure time(Table IV).

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Total Pb concentrations in plant tissues,earthworms,and soil samples

It was clearly observed that the concentration of Pb was higher in roots than in shoots of the plants(Fig.8).However,the concentration of Pb in the plants from the P+E set was less when compared with that of the plant from the P+E+B set.This indicates that the combined strategy was more efficient in Pb uptake,thus proving to be a more efficient system for Pb bioremoval.

The earthworm samples also showed accumulation of Pb in its biomass.The concentration of Pb in the earthworm tissues increased with increasing Pb concentration in the soil(P＜0.001).In the P+E+B set,the accumulation of Pb in the earthworms was very high when compared with that of the earthworms in the P+E set.Figure 9 represents the bioaccumulation factor for Pb in the biomass of earthworms.

根据国际能源署（IEA）的最新月报，11月份欧佩克原油产量环比进一步提高10万桶/日，达到3303万桶/日。增产主要来自沙特阿拉伯和阿联酋，这两个国家原油产量环比分别提高41万桶/日和11万桶/日，分别达到1106万桶/日和333万桶/日的最高纪录。伊朗原油产量环比减少31万桶/日，至301万桶/日，为2016年以来最低水平，相比2017年同期减少84万桶/日。伊朗石油出口量环比减少55万桶/日，至128万桶/日，自5月份以来已减少140万桶/日。

Figure 8 presents the concentration of Pb in the soil with and without LRB.The concentration of Pb in the soil reduced more significantly in the P+E+B set when compared with that of the P+E set.

Fig.7 Locomotion of the earthworms,expressed as time taken to cover the distance of 30 cm,in the experimental pots of two sets(P+E and P+E+B)artificially contaminated with different Pb concentrations.The locomotion was determined based on the distance covered by the earthworms in 1 min(Lemtiri et al.,2016).Vertical bars are the standard deviations of means.Control=without Pb supplementation;P+E set=plants augmented with earthworms;P+E+B set=plants augmented with earthworms and lead-resistant bacterium.dw=dry weight.

TABLE III Number of colonies of the gut microbes of the earthworms in the experimental pots of two sets(P+E and P+E+B)artificially contaminated with different Pb concentrationsa)

a)Control=without Pb supplementation;P+E set=plants augmented with earthworms;P+E+B set=plants augmented with earthworms and Pb-resistant bacterium. b)Dry weight. c)Colony-forming units.

Pb concentration P+E P+E+B mg kg−1dwb)CFUc)Control ＞700 ＞1000 50 490 790 100 120 340 150 92 180

TABLE IV Malondialdehyde(MDA)content of earthworms in the experimental pots of two sets(P+E and P+E+B)artificially contaminated with different Pb concentrationsa)

a)Control=without Pb supplementation;P+E set=plants augmented with earthworms;P+E+B set=plants augmented with earthworms and Pb-resistant bacterium. b)Dry weight.

Pb concentration P+E P+E+B mg kg−1dwb)µmol g−1 Control 10.0 6.6 50 12.5 8.5 100 15.6 10.5 150 17.5 11.0

Fig.8 Concentrations of Pb in plant tissues(roots and shoots)and soil in the experimental pots of two sets(P+E and P+E+B)artificially contaminated with different Pb concentrations of 50,100,and 150 mg kg−1dry weight(dw),measured on Days 30(a)and 60(b)of growth.Vertical bars are the standard deviations of means.Control=without Pb supplementation;P+E set=plants augmented with earthworms;P+E+B set=plants augmented with earthworms and Pb-resistant bacterium.

Fig.9 Bioaccumulation factor of Pb in body mass of the earthworms in the experimental pots of two sets(P+E and P+E+B)artificially contaminated with different Pb concentrations.Vertical bars are the standard deviations of means.Control=without Pb supplementation;P+E set=plants augmented with earthworms;P+E+B set=plants augmented with earthworms and Pb-resistant bacterium.

DISCUSSION

Different ecotypes with variable abilities to accumulate Pb have been reported from around the world(Sol´ıs-Dom´ınguez et al.,2007;Khan et al.,2014).Several investigations have been performed to reveal the uptake of heavy metals with a consortium of plants and bacteria(Dhawi et al.,2016),plants and earthworms(Lemtiri et al.,2016),and earthworms and bacteria(Jusselme et al.,2012).The present study used all the three living systems,viz.,plants,earthworms,and bacteria,for the enhanced uptake of heavy metal,especially Pb.To the best of our knowledge,the present study is the first to use all the three living systems to remediate Pb.

Lead-resistant bacteria were isolated from the contaminated sites near tannery industries in Erode District.An understanding of the soil characteristics is necessary for the study of heavy metal uptake from contaminated soil and its use in pot culture.In the present study,the complete soil pro filing was performed at National Agro Foundation.The organic content of the soil is one of the key factors driving the microbial community structure.Soil with high heavy metal content had a high organic content,which can probably explain the maintenance of the microbial community diversity due to lack of competition(Pouyat et al.,2015).Furthermore,this enabled the isolation of effective LRB from the contaminated soil,which was able to tolerate high Pb concentration of up to 1000 mg L−1(VITMVCJ1)in the present study.Based on the morphological and biochemical characteristics,comparative analysis of 16S rRNA gene sequence,and phylogenic analysis,the strain was identified to be homologous to Klebsiella variicola(99.9%similarity).Klebsiella variicola has already been reported to possess a significant potential to remove Pb from the soil(Mu˜noz et al.,2015).Therefore,the bacterial strain isolated in the present study might possess the potential for the safe treatment of Pb-contaminated water and soil.Furthermore,the Pb-resistant gene,pbrA,was identified in VITMVCJ1.The gene has been previously identified in Ralstonia metallidurans as playing an active role in the uptake,efflux,and accumulation of Pb(Borremans et al.,2011).Thus,the isolate VITMVCJ1 was employed in the experiment of the present study.

A hyperaccumulator plant P.purpureum,a perennial grass,was selected as a tool for bioremediation.The fact that P.purpureum biomass continuously increased and no plant mortality was observed in the present study indicated that the plant was able to survive in the Pb-contaminated soil,which is essential for phytoremediation.Fast growth,high above ground biomass yield,and rich root system of plants are the requisites for phytoextraction purposes.The results of the present study were consistent with the findings of Mant et al.(2006),who found that P.purpureum exposed to Cr(III)showed a metal removal efficiency of 78%.Lumbricus terrestris was selected as a model earthworm to study the variation in toxic effects of Pb with LRB augmentation.Previous reports on Pb bioaccumulation by L.terretris reveal that the increase in the concentration of Pb has toxic effects on earthworms(ˇZaltauskait˙e and Sodien˙e,2010).

Our field survey showed that P.purpureum was hyper-tolerant to Pb and,when accompanied with LRB and earthworms(P+E+B),grew healthily on contaminated soil with up to 150 mg kg−1Pb.It was observed that Pb significantly retarded the growth attributes,including the root and shoot lengths of P.purpureum.Furthermore,increasing effects were noticed in plants with higher Pb concentration without bioaugmentation.This is in agreement with the study conducted by Khan et al.(2014).However,bioaugmentation with Klebsiella sp.had a positive effect on the production of plant biomass.Furthermore,LRB might have enhanced the host plant growth by inducing the biochemical pathways of the plant to produce phytohormones or by increasing the availability of nutritional elements for the host plant.There was a statistically significant decrease in chlorophyll content in the plants after exposure to the Pb-contaminated soil.Interestingly,with bioaugmentation,the chlorophyll content gradually increased up to levels greater than the initi-al values.The content of chlorophyll in the leaves of plants is a marker to estimate the photochemical activity,thus revealing plant stress(Cordon et al.,2016).It is a useful parameter to assess plant health.Flavonoids are secondary metabolites of plants,the antioxidant activity of which depends on the presence of free hydroxyl groups(Geetha et al.,2003).Phenolic compounds have free radical scavenging ability and could be used for rapid screening of antioxidant activity(Soobrattee et al.,2005).In the present study,the antioxidant activity exhibited a positive correlation(R2=0.99)with the phenolic content of the plant.This con firms that the phenolic content of plants contributes directly to their antioxidant properties.The higher phenolic and flavonoid contents were found in the set without bioaugmentation.Therefore,in response to oxidative stress,plants were able to develop antioxidant defense systems,thus comprising the synthesis of protective compounds with antioxidant activity(Gill and Tuteja,2010).Besides,roots of plants from the P+E+B set were found to be strong bio film producers,which enhanced uptake of Pb by the root.Generally,bacterial cells produce signal molecules,which allow the entire population to expand as a bio film over the root surface(Ullah et al.,2015).

In the present study,the presence or absence of LRB significantly affected the toxic effects of Pb on earthworms.Earthworms represent a significant proportion of the soil biomass and are regarded as useful indicators of soil quality and health(Edwards et al.,2004).The P+E set presented some toxic effects on earthworms at the morphology level,including its locomotion efficiency.Furthermore,the body weight of earthworms decreased with increase in the concentration of Pb and exposure time.However,in the P+E+B set(LRB-augmented)of the present study,the survival rate of earthworms was greatly enhanced.The lack of mortality of biological organisms in old contaminated soils was reported by Schreck et al.(2011).The present study showed alterations in the midgut tissue of earthworms.There was a drastic decline in the enumeration of gut microbes as the concentration of Pb increased in the P+E setup(without LRB).When augmented with LRB,a better survival of the earthworms was observed due to the bacterial uptake of Pb.The Pb-resistant nature of the supplemented bacteria might enrich the micro flora lost due to Pb toxicity,thus helping combat the stress associated with Pb exposure.Reports have proved that the gut micro flora of earthworm is important for various life processes and therefore for its survival(Chen et al.,2016).The Pb-stress condition on earthworms can be alleviated by the decrease in MDA content.Malondialdehyde is a secondary end product of the oxidation of polyunsaturated fatty acids(Del Rio et al.,2005)and is used as an index of general lipid peroxidation(Li et al.,2015).The highest levels of MDA content were found in earthworms in the P+E set.

Bioaugmentation of soil with Klebsiella sp.had a statistically significant effect on Pb concentration in the root and shoot of the plants and also on the body mass of earthworms.The present study also investigated the effect of L.terrestris on the ability of the plant to take up Pb in contaminated soils.The concentration of Pb was found to be higher in the shoot and root of P.purpureum growing in bioaugmented soil along with earthworms.Pennisetum purpureum appeared to be a good candidate for Pb extraction.Furthermore,the AAS result validated that the setup with all the three living biosystems(P+E+B)showed more Pb uptake,30%more effective when compared with that of the setup without bioaugmentation.

CONCLUSIONS

The bioaugmentation of microbes with an association of living biosystems(plants and earthworms)proved efficacious in the removal of Pb from the contaminated soil.The concentration of Pb accumulated by the integrated biosystems using plant,earthworms,and bacteria was found to be 87 mg kg−1,which was 30%higher than that of the non-integrated system.The toxic effects of Pb on plants and earthworms were also found to be reduced upon bioaugmentation with effective bacteria that aided to increase the micro flora in both plant rhizosphere and earthworms,indicating the enrichment of the microbe population lost with increasing Pb concentrations.This signifies the role of LRB in Pb tolerance.The study also led to the development of better in situ remediation strategies and a better understanding of the interaction between earthworms,plant,and bacteria for enhanced bioremediation of Pb in a novel manner,and thus can be carried out on a vast scale.From the comparison between combined and single processes employed to bioremediate polluted soils,it can be concluded that combining multiple bioremediation approaches could enhance the removal of contaminants from the soil.However,further research is needed to optimize the species combinations for suitable heavy metal uptake.

ACKNOWLEDGEMENTS

We are grateful to the management of the farm in Brahmapuram,India for providing the plants.We are thankful to the management of the farm in Bagayam,Vellore,India for providing the earthworms needed for the study.We extend our appreciation to the National Agro Foundation,Chennai,India for analyzing the soil.We extend our heartfelt gratitude to the nursery of the CBMR,Vellore Institute of Technology,India for providing the space for carrying out the study.We are grateful to the TBI facilities at Vellore Institute of Technology,India for carrying out the AAS analysis of the samples.We are thankful to the management of Vellore Institute of Technology,India for providing the lab facilities for conducting the study.

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