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Applied Soil Ecology 62 (2012) 142– 146

Contents lists available at SciVerse ScienceDirect

Applied Soil Ecology

journa l h o me page: www.elsev ier .com/ locate /apsoi l

hort communication

ssessment of Miscanthus × giganteus secondary root metabolites for theiostimulation of PAH-utilizing soil bacteria

idier Techer ∗, Marielle D’Innocenzo, Philippe Laval-Gilly, Sonia Henry, Amar Bennasroune,laudia Martinez-Chois, Jairo Falla

niversité de Lorraine, Laboratoire des Interactions Ecotoxicologie, Biodiversité, Ecosystèmes (LIEBE), CNRS UMR 7146, IUT Thionville-Yutz, Espace Cormontaigne, Yutz, F-57970,rance

r t i c l e i n f o

rticle history:eceived 31 October 2011eceived in revised form 28 April 2012ccepted 5 June 2012

eywords:

a b s t r a c t

The objective of this study was to identify secondary root metabolites of Miscanthus × giganteus thatcould be involved in the biostimulation of PAH-utilizing soil bacteria. The use of such compoundsin situ could eventually help enhance the bioremediation of various contaminated sites. First, anenrichment for a PAH-degrading mixed bacterial culture was conducted using pyrene and phenan-threne as carbon sources. Then, the biomass production and catabolic activity of the mixed bacterial

iscanthus × giganteusecondary root metabolitesioremediationAHuercetin

culture were evaluated in the presence of Miscanthus × giganteus root exudate and PAH. For this pur-pose, spectrophotometric measurements were performed using microplate assays. The results indicatethat the addition of root exudate promoted bacterial growth and the catabolic activity of poly-cyclic molecules. Additional assays with selected secondary root metabolites (quercetin, catechin,apigenin and gallic acid) identified quercetin as the major contributor to the bacterial biostimulationprocesses.

. Introduction

Bacterial degradation of recalcitrant organic contaminants, suchs polycyclic aromatic hydrocarbon (PAH), is often stimulated inhizosphere soils (Aprill and Sims, 1990; Binet et al., 2000; Daanet al., 2001; Euliss et al., 2008; Fletcher and Hegde, 1995; Germidat al., 2002; Gilbert and Crowley, 1997; Ma et al., 2010; Miya andirestone, 2001; Singer et al., 2003; Yi and Crowley, 2007). Thishenomenon has been largely attributed to the secretion of rootxudates: a complex mixture of sugars, amino acids, organic acidsnd phenols released in the vicinity of plant roots (Aprill and Sims,990; Badri et al., 2009; Binet et al., 2000; Fletcher and Hegde,995; Germida et al., 2002; Gilbert and Crowley, 1997; Ma et al.,010). These compounds may enhance microbial communities andatabolic activities 5–100-fold in the root zone compared to bulk

oil (Germida et al., 2002).

Enzymatically, the initial oxidations of PAH generally involveubstrate-specific dioxygenases that incorporate two oxygen

Abbreviations: PAH, polycyclic aromatic hydrocarbon; PCB, polychlorobiphenyl;EPG, yeast extract peptone glucose.∗ Corresponding author at: Laboratoire des interactions écotoxicologie, biodiver-

ité, 25 ecosystèmes (LIEBE), CNRS UMR7146, Université Metz Paul Verlaine, IUThionville-Yutz, Impasse A. Kastler, 57970 Yutz, France. Tel.: +33 6 13 07 92 41.

E-mail address: erwann9@hotmail.com (D. Techer).

929-1393/$ – see front matter © 2012 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.apsoil.2012.06.009

© 2012 Elsevier B.V. All rights reserved.

atoms into an aromatic ring (Cerniglia, 1992; Haritash and Kaushik,2009; Shuttleworth and Cerniglia, 1995). These enzymes, whichvary according to the substrate and bacterial strain, can havebroad substrate specificity (Cerniglia, 1992; Rentz et al., 2004). Forexample, naphthalene dioxygenases are versatile enzymes that cancatalyze the initial oxidations of naphthalene, as well as phenan-threne, fluorene and anthracene (Resnick et al., 1996; Sanseverinoet al., 1993). Like most enzymes, PAH-dioxygenase synthesismay be induced to high levels by either a pathway substrateor intermediate (Cerniglia, 1992; Díaz and Prieto, 2000). Indeed,Chen and Aitken (1998) demonstrated that the pre-incubation ofPseudomonas saccharophila P15 strain with phenanthrene or sal-icylate (an intermediate metabolite found in the naphthalene-and phenanthrene-degrading pathways) both stimulated dioxyge-nase activity and increased rates of pyrene removal. Furthermore,some structural analogues of PAH and their intermediate metabo-lites could be considered as “fortuitous” inducers because theyinduce biodegradation pathways for specific pollutants in soils(Díaz and Prieto, 2000; Yi and Crowley, 2007). Therefore, manyresearchers have speculated that some categories of secondaryplant metabolites could act as natural promoters of organic pol-lutant degradation in rhizosphere soils.

Miscanthus × giganteus is a sterile perennial C4 Gramineae,which is able to grow in various low quality, polluted soils with-out any nutrient input (Fernando et al., 2004; Lewandowski et al.,2003). These agronomically interesting traits suggest there are

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ntensive interactions among the plant, soil and microorganismshrough the secretion of various secondary root metabolites thatventually ensure the availability of nutrient supplies and resis-ance to multiple stressors (Badri et al., 2009; Leung et al., 2007).

This study aimed to identify the secondary metabolites in Mis-anthus × giganteus root exudates that might act as “fortuitous”romoters of PAH biodegradation through the biostimulation ofAH-utilizing bacteria. Such phytochemicals may enhance theatabolic activity (and cellular growth) of bacteria, thereby result-ng in pollutant degradation and, ultimately, dissipation. Thepplication of these compounds to contaminated sites could proveo be a practical and innovative bioremediation approach, poten-ially replacing time-consuming phytoremediation and other costlyhysicochemical techniques.

In the current study, a PAH-degrading mixed bacterial cultureas enriched from industrially contaminated soil. Miscant-

us × giganteus total root exudate was tested for biostimulationf the mixed bacterial culture using a rapid microplate assay.hen, to thoroughly investigate the biostimulation of the PAH-tilizing bacteria by the root exudates, additional in vitro assaysere conducted with four of the major secondary root metabolites

f Miscanthus × giganteus: quercetin, apigenin, catechin and galliccid.

. Materials and methods

.1. Isolation of a PAH-degrading mixed bacterial culture

A soil sample characterized by a total PAH contamination of480 mg kg−1 dry soil (with a pyrene concentration of 450 mg kg−1

ry soil) was collected from a former coke industry site fromhe North East of France. A bacterial inoculum was prepared by

ixing 10 g of this soil with 50 ml of a buffered saline solu-ion for 10 min (Gaskin and Bentham, 2005). Ten milliliters ofhis inoculum was added to a selective enrichment flask contain-ng a mineral medium supplemented with 200 mg l−1 pyrene and00 mg l−1 phenanthrene (Gaskin and Bentham, 2005). Pyrene is

well-known recalcitrant four-ring PAH, whereas phenanthrenes a three-ring hydrocarbon that can enhance the expression ofioxygenases and subsequent cometabolism of recalcitrant com-ounds (Gaskin and Bentham, 2005; Johnsen and Karlson, 2007).trains from the mixed bacterial culture were isolated on non-elective yeast extract peptone glucose (YEPG) media containing

g l−1 yeast extract, 10 g l−1 peptone and 10 g l−1 glucose. Theseacteria were then transferred to pyrene-sprayed agar platesEuliss et al., 2008) to confirm their ability to degrade pyreneTable 1). The nucleic acids were extracted from distinctively iso-ated colonies and used for PCR amplification of the 16S rDNA.niversal primers targeting the hypervariable V3 region weresed to amplify fragments of 200 bp (corresponding to positions41–534 in the Escherichia coli sequence) (Muyzer et al., 1993). TheCR products were sequenced directly (GENOSCREEN) using theforementioned universal primers. The best matching sequence inhe public domain of the Ribosomal Database Project (RDP) wasdentified with the program SIMILARITY RANK (Cole et al., 2006).t should be noted that, although the RDP matches were, in someases, to bacteria named to the species or strain level (Table 1),he proper assignment of the isolates to species would requiredditional tests. In the absence of these data, all interpretationsegarding bacteria isolated in the present work were limited to theenus level.

.2. Root exudate collection

Rhizomes of Miscanthus × giganteus were grown in quartz sandor one month in a phytotronic chamber (7000 lux, 16/8 h, 24 ◦C).

logy 62 (2012) 142– 146 143

Root exudates were collected by first immersing the roots in500 ml of sterile, distilled water for 2 h and then concentrating theexudates by lyophilization. A solution of 10 mg ml−1 of root secret-ions was prepared in sterile, distilled water for use in microplateassays.

2.3. Design of microplate assays

Bacterial biostimulation assays were conducted using 96-wellmicrotiter plates. First, the biostimulation effects of Miscant-hus × giganteus total root exudates on the mixed bacterial culturewere investigated. For this purpose, the wells were divided into fourcategories based on the addition of PAH (10 �l), exudate (1.4 �l)or PAH (10 �l) + exudate (1.4 �l) or control wells (containinggrowth medium only). The PAH consisted of pyrene, fluoran-thene, fluorene and phenanthrene dissolved at concentrations of1.1/1.1/1.1/11.1 mg l−1 in hexane, respectively (Binet et al., 2000).The solvent was allowed to evaporate overnight under a fumehood. Subsequently, 200 �l of a ten-fold dilution of YEPG growthmedium and 25 �l of the mixed bacterial culture (107 cells ml−1)were dispensed into each well. The bacterial growth was measuredspectrophotometrically as the O.D620 nm after 6 days of incubation.The catabolic activity was estimated by measuring the formation ofintermediate metabolites, which was determined by the O.D405 nmminus the O.D620 nm (O.D405–620 nm) (Binet et al., 2000).

The four major secondary metabolites contained in thepolyphenolic fraction of Miscanthus × giganteus root exudates, i.e.,quercetin, catechin, apigenin and gallic acid (identified by HPLCprior to this work, Técher et al., 2011), were tested in additionalbacteria biostimulation assays. Methanol suspensions of each ofthese secondary metabolites (purchased from Sigma–Aldrich) wereprepared at a concentration of 17.22 mM (corresponding to a cat-echin suspension of 5 mg ml−1). The suspensions were mixed andthen added into the wells (1.4 �l per well) according to a full facto-rial design comprising 32 treatments: 25 combinations based on thepresence/absence of the four secondary metabolites and PAH. Thesame amount of methanol was also added to the control medium(YEPG without PAH). The bacterial growth and catabolic activitywere measured spectrophotometrically as previously described.

2.4. Statistical analyses

All data were expressed as the mean ± SD. The differencesbetween groups were determined by ANOVAs for parametricdistributions or Kruskal–Wallis for non-parametric distributionsfollowed by Student Newman–Keuls post hoc tests (p < 0.05). Theanalyses were performed using SigmaStat statistical software (v.3.5, Systat Software Inc., Point Richmond, California).

3. Results and discussion

3.1. Isolation of a PAH-utilizing mixed bacterial culture

It has been well documented that the use of liquid mediumusually favors the growth of “r” strategist bacterial strains. Suchmicroorganisms are characterized by a rapid growth rate and areoften represented by Gram-negative bacteria belonging to the phy-lum of proteobacteria (Bernard et al., 2007; Fierer and Jackson,2006). Similar observations were made in the present work witheleven isolated strains, which belonged to ˇ- or �-proteobacteria(Table 1). Moreover, all of the identified bacterial genera (Table 1)have been previously reported as PAH-degraders in the literature

(Daane et al., 2001; Germida et al., 2002; Johnsen et al., 2005).Among them, the Pseudomonas and Stenotrophomonas genera havebeen cited in several studies for their degradation of organic com-pounds (including xenobiotics) in the rhizosphere (Boonchan et al.,

144 D. Techer et al. / Applied Soil Ecology 62 (2012) 142– 146

Table 1Identification of the bacterial isolates obtained from the PAH-degrading mixed culture after enrichment with pyrene + phenanthrene, as determined by 16S rDNA analysis.

Phylogenetic affiliation S ab scorea RDP accession numberb Closest homologous sequencec Pyrene-degrading capacityd

proteobacteria 1.000 S000394481 Delftia acidovorans +� proteobacteria 1.000 S000511878 Bacterium QLW23-isolateX (Rheinheimera) +� proteobacteria 1.000 S000030774 Stenotrophomonas sp. DFK5 +� proteobacteria 1.000 S001292390 Pseudomonas sp. AKB-2008-E116 ++� proteobacteria 0.971 S001055855 Uncultured Citrobacter sp. ++� proteobacteria 0.965 S000877284 Uncultured Pantoea sp. +� proteobacteria 1.000 S000134506 Pseudomonas mendocina +� proteobacteria 0.978 S000040856 Potato plant root bacterium RC-III-94 (Enterobacteriaceae) ++� proteobacteria 1.000 S000363818 Uncultured gamma proteobacterium Pseudomonas sp. ++ˇ proteobacteria 1.000 S000111909 Delftia acidovorans +� proteobacteria 1.000 S001046872 Pseudomonas sp. 01WB02.2-23 ++

a Similarity coefficient for query and matching sequences (Cole et al., 2006).atabas

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b Accession number of the closest homologous sequence referenced in the RDP dc Nearest homolog in the RDP database (Cole et al., 2006).d ++ growth of isolate observed on a pyrene-sprayed agar plate with a clear zone

998; Daane et al., 2001; Johnsen et al., 2005; Rojo, 2010). Thus,he strains found in the mixed bacterial culture were capable ofeacting quickly to the addition of Miscanthus × giganteus root exu-ates and the derived components during the microplate assays.

.2. Effects of total root exudate on bacteria biostimulation

The mixed bacterial culture showed a marked catabolic activityollowing the addition of Miscanthus × giganteus total root exu-ates (Fig. 1B). This result was correlated with the assimilation ofhe plant-derived compounds by the mixed culture because thereas an average OD620 of 0.27 in the presence of the root exu-ates compared with that of 0.20 for the YEPG control mediumFig. 1A). The significant growth (Fig. 1A) and catabolic activity

Fig. 1B) increases exhibited by the bacterial culture in the pres-nce of PAH also confirmed the efficiency of PAH metabolism andhe use of PAH as a carbon and energy source by the culture. Theigher catabolic activity observed with the combination of PAH

ig. 1. Assessment of the addition of PAH, root exudates (+Ex) and the combinationf both (+PAH + Ex) on: (A) the biomass production (O.D620 nm) and (B) the rela-ive catabolic activity (O.D405–620 nm) of the PAH-degrading mixed bacterial culturen yeast extract peptone glucose (YEPG) medium (mean of four replicates ± SD);he capital letters indicate a significant difference determined by 2-way ANOVAp < 0.05).

e.

unding colonies; + growth of isolate observed without any clear zone.

and root exudates (Fig. 1B) suggested there was an enhancementof PAH biotransformation with the root exudates with presumablymore assimilative biodegradation of pollutants in this experimentalcondition (Fig. 1A).

3.3. Effects of secondary root metabolites on bacteriabiostimulation

To confirm the presence of “fortuitous” inducers of PAHbiodegradation in Miscanthus × giganteus root exudates, additionalbiostimulation assays were conducted with gallic acid, catechin,apigenin and quercetin (Figs. 1 and 2). These compounds are foundin the mixture of secondary root metabolites naturally secretedby Miscanthus × giganteus. In the absence of PAH (Fig. 2A, whitecolumns), the addition of secondary metabolites to the growthmedium induced only minor variations in the OD620 compared tothe average value in the control medium, i.e., YEPG only (Fig. 2A,col. 1). Indeed, slight increases in bacterial biomass were noted for afew polyphenol combinations (Fig. 2A, white columns: col. 4, 6, 7, 8,15 and 16, all marked with “*”). The measurement of the OD405–620confirmed that the contribution of these compounds to the globalcatabolic activity measured in bacterial cultures was minor (Fig. 2B,white columns: 4, 6, 7, 8, 10, 12, 13, 15 and 16, all markedwith “*”).

The bacterial cultures experienced significant growth increasesin the presence of PAH compared to growth in the control mediumYEPG alone (Fig. 2A, all colored columns, marked with “*”). Theassimilation of PAH by biodegradation was confirmed by mea-suring increases in the catabolic activity of the bacterial culturein the presence of pollutants (except for catechin + PAH; Fig. 2B,all colored columns). Moreover, the bacterial growth was clearlyenhanced in all of the treatments that had the simultaneouspresence of quercetin and PAH (Fig. 2A, colored columns from 9 to16), with the highest OD620 values measured for bacterial culturesin the presence of PAH plus quercetin plus gallic acid (Fig. 2A,colored column 10), followed by PAH plus quercetin (Fig. 2A,colored column 9). It must be noted that the addition of quercetinalone to the YEPG medium was not associated with either asignificant increase in bacterial growth (Fig. 2A, white column 9)or catabolic activity (Fig. 2B, white column: 9). In other words, inour experimental conditions, quercetin alone was not sufficient tosupport significant bacterial biomass production. In addition, theanalysis of catabolic activity for all culture conditions involvingPAH and quercetin showed significant increases when compared

to the corresponding culture condition without pollutants (Fig. 2B,colored columns from 9 to 16, marked with “a”). This result clearlysuggests that the biodegradation of organic contaminants andtheir use as a carbon and energy source for bacterial biomass

D. Techer et al. / Applied Soil Ecology 62 (2012) 142– 146 145

Fig. 2. Assessment of the addition of the secondary plant metabolites gallic acid (+A.G.), catechin (+Cat.), apigenin (+Api.) and quercetin (+Quer.), PAH and their combinationon: (A) the bacterial growth and (B) the relative catabolic activity (O.D.405–620 nm) of the PAH-degrading mixed bacterial culture (mean of six replicates ± SD). Treatments arenumbered from 1 to 16. For each numbered treatment, white columns indicate the absence of PAH (−PAH) in the growth medium whereas orange columns indicate thep ne glus gnific(

phasGobtrwsAlaitessoa

resence of PAH (+PAH). The first treatment “YEPG” stands for yeast extract peptoignificant difference versus the first treatment YEPG only (white column) and # a siwhite column) for each treatment (p < 0.05).

roduction occurs in the presence of quercetin. These results alsoighlighted the role of quercetin in the stimulation of the catabolicctivity of polyaromatic compounds in the presence of PAH withubsequent bacterial growth. Similar results were obtained byilbert and Crowley (1997), who demonstrated the inductionf polychlorinated biphenyl (PCB) degradation in Gram-positiveacteria following the addition of spearmint root products tohe growth medium. L-Carvone was identified as the compoundesponsible for this phenomenon. Interestingly, this compoundas not a growth substrate for bacteria, and even showed bacterio-

tatic effects in presence of fructose (Gilbert and Crowley, 1997).ccording to the authors, the absorption of L-carvone presumably

ed to the induction of detoxification metabolic pathways, whichre also involved in PCB catabolism. Taking this informationnto account, it is possible that quercetin may act similarly inhe biostimulation of PAH-utilizing bacteria. Indeed, despite thevolutionary adaptation of aromatic ring-degrading enzymes for

pecific substrates, the enzymes for a particular pathway canhow less specificity and catalyze the transformation of a range ofther aromatic compounds (Díaz and Prieto, 2000; Shuttleworthnd Cerniglia, 1995; Yi and Crowley, 2007). Extrapolating from

cose (the growth medium) without any secondary plant metabolite. * indicates aant difference between each condition with PAH (orange column) and without PAH

this concept, another substrate may be ‘accidentally’ transformedbecause of structural similarities with the primary substrate.Therefore, the oxidation and hydroxylation processes involved inthe detoxification of the secondary plant metabolite quercetin mayalso occur for PAH, making the resulting molecule more solubleand reactive in further biochemical reactions associated with itsbiodegradation. Indeed, the initial intracellular attack of organicpollutants like PAH is an oxidative process where the activation andincorporation of oxygen is the key enzymatic reaction catalyzed byoxygenases. The enzymes used for the biodegradation of PAHs canbe classified in two groups: the peripheral and the fission enzymes.The peripheral enzymes are involved in the recognition of PAHand their biotransformation into more reactive and degradablemolecules (Mishra et al., 2001). The enzymes involve in the bio-transformation of quercetin may also act on the initial steps of PAHoxidation. The fission enzymes would then pass the resulting PAHintermediate metabolites through the common routes of energy

generation and carbon rout for assimilation by the microbial cell(Mishra et al., 2001). Finally, the lower hydrophobicity of quercetincompared with that of PAH may enhance its cell absorption andshorten the time required for the induction of enzymes involved in

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he detoxification processes, leading ultimately to the stimulationf xenobiotic biotransformation.

. Conclusion

These in vitro assays showed the potential of Miscant-us × giganteus root exudates to promote the growth and catabolicctivity of PAH-utilizing bacteria. Further container experimentsnd field studies are needed to confirm the phytoremediationffects of this grass in situ. The current study also indicated that theddition of the secondary root metabolite quercetin could enhancehe degradation activity of PAH-utilizing bacteria in the presencef pollutants, resulting in increased microbial growth. Such find-ngs may pave the way for the development of direct remediationpproaches for PAH polluted environments with no need for plant-ng or using other costly physicochemical techniques.

cknowledgments

This study was partially supported by the French govern-ental agency ADEME (Agence de l’Environnement et de laaîtrise de l’Energie), EDF (Electricite De France) and Commu-

auté d’agglomérations Porte de France, Thionville. We would likeo thank also Marielle D’Innocenzo for her technical assistance.

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