Isolation and Characterization of Hexavalent Chromium-Reducing Rhizospheric Bacteria From a Wetland

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Isolation and Characterization of Hexavalent Chromium-ReducingRhizospheric Bacteria From a WetlandAmparo Mauricio Gutiérrez a; Juan José Peña Cabriales a;María Maldonado Vega b

a Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional CampusGuanajuato, Irapuato, Guanajuato, México b Dirección en Investigación Ambiental, Centro deInnovación Aplicada en Tecnologías Competitivas A. C., León, Guanajuato, México

Accepted uncorrected manuscript posted online: 29 January 2010

First published on: 29 January 2010

To cite this Article Gutiérrez, Amparo Mauricio , Cabriales, Juan José Peña andVega, María Maldonado(2010) 'Isolationand Characterization of Hexavalent Chromium-Reducing Rhizospheric Bacteria From a Wetland', International Journalof Phytoremediation, 12: 4, 317 — 334, First published on: 29 January 2010 (iFirst)To link to this Article: DOI: 10.1080/15226510902968118URL: http://dx.doi.org/10.1080/15226510902968118

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International Journal of Phytoremediation, 12:317–334, 2010Copyright C© Taylor & Francis Group, LLCISSN: 1522-6514 print / 1549-7879 onlineDOI: 10.1080/15226510902968118

ISOLATION AND CHARACTERIZATION OF HEXAVALENTCHROMIUM-REDUCING RHIZOSPHERIC BACTERIAFROM A WETLAND

Amparo Mauricio Gutierrez,1 Juan Jose Pena Cabriales,1

and Marıa Maldonado Vega2

1Centro de Investigacion y de Estudios Avanzados del Instituto PolitecnicoNacional Campus Guanajuato, Irapuato Guanajuato, Mexico2Direccion en Investigacion Ambiental, Centro de Innovacion Aplicada enTecnologıas Competitivas A. C., Leon Guanajuato, Mexico

Scirpus americanus Pers. occurs naturally in “San German,” a pond that serves as a recep-tor of industrial wastewater in Guanajuato, Mexico. This plant accumulates metals mainlyin the root: concentrations (mg/kg) of Cr, As, Cd and Se were 970, 49, 41, and 85 respectively.Analysis of rhizosphere samples indicated bacterial population of 108 cfu g−1 in media with0.2 mM Cr(VI) and 10 mM sodium gluconate. Thirteen isolates were obtained and phy-logenetic analyses (16S rRNA) indicated they corresponded to genera of Agrobacterium,Arthrobacter, Microbacterium, Curtobacterium, Rhodococcus, Xanthomonas and Pseu-domonas. Cr(VI) reduction was evaluated using the diphenyl carbazide method. The isolatesaccomplished 5–40% (20 µM) of reduction in assays of resting cell and tolerated 0.5–5.0mM Cr(VI). Eight strains used nitrate and thirteen used iron and chromium as electronacceptors to grow under anaerobic conditions. Cr(VI) reduction by five strains occurred atpH values (7–9) and NaCl concentrations (0.5–1.0 M) in basal medium. A mixed culture ofstrains (S17 and S28) reached a chromium removal of 100% at 0.2 mM Cr(VI) initial con-centration. Aerobically, this consortium was capable of 93.8% Cr(VI) reduction of 81 µgL−1

Cr(VI) of the industrial effluent, indicating their possible use in environmental cleanup.

KEY WORDS Bioremediation, bacterial consortia, Scirpus americanus Pers

INTRODUCTION

Leon Gto. Mexico (Central Mexico) is a city with intense industrial activity. The vastmajority of Leon’s industries do not treat their effluents. The wastewaters are dischargeddirectly into either the local river Turbio (part of one of Mexico’s most important riverbasins, the Lerma-Chapala), or into lagoons and ponds. “San German” is one such pond thatserves as a receptor of industrial wastewater. This wastewater contains high concentrationsof heavy metals (chromium), sulfates and organic matter (Armienta and Rodrıguez, 1995;Armienta et al., 1996).

Address correspondence to Juan Jose Pena Cabriales, Centro de Investigacion y de Estudios Avanzados delInstituto Politecnico Nacional Campus Guanajuato, Km. 9.6 Libramiento Norte, Carretera Irapuato-Leon Apdo.postal 629 C.P. 36500, Irapuato Guanajuato, Mexico. E-mail: [email protected]

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318 A. M. GUTIERREZ ET AL.

The sources of chromium are leather tanning, electroplating, pigmentation, and metalfinishing. Wastewaters containing Cr(VI) from these industries represent a well recognizedbio-hazard (Losi et al., 1994; Kimbrough et al., 1999). Cr(VI) and Cr(III) are the moststable oxidation states of the element. The first is toxic, soluble and mobile in aquatic andterrestrial environments (Kozuh et al., 2000). Cr(VI) is genotoxic and cytotoxic for bacteriaand eukaryotic cells, also it is a known carcinogen and mutagen agent of mammals (De Floraet al., 1990; Snow, 1992; Costa, 1997; Shrivastava et al., 2005). Cr(III), on the other hand, isstable and an essential element for nutrition. For human it is recommended adequate dailydietary intake of 50–200 µg/day (Barceloux, 1999; Anderson, 2000) although at higherconcentrations it can enter cells and reach toxic levels intracellularly (Beyersmann andHartwig, 2008).

Conventional methods for removing Cr(VI) ions from industrial wastewater includereduction, reduction followed by chemical precipitation, biosorption by coating chitosan onalumina, reverse osmosis, ion-exchange and electrolytic methods (Petruzzelli et al., 1995;Boddu et al., 2003; Fathima et al., 2005; Lu et al., 2006). These methods are expensivebecause they often involve high capital and operational costs.

Phytoremediation is a biotechnology cheaper than conventional remediation methods.The use of plants to extract, sequester, and detoxify pollutants has been reported to beeffective to remediate polluted systems, and in some cases, it might become the preferredtechnology (Cunningham and Ow, 1996). Different plants have been identified for theircapacity to bioconcentrate chromium in their root system: Thlaspi caerulescens J.Presl& C.Presl (Baker et al., 1994), Scirpus lacustris L. (Suseela et al., 2002), Convolvulusarvensis L. (Gardea-Torresdey et al., 2004), Helianthus annuus L. (Turgut et al., 2004),Brassica juncea L. (Bluskov et al., 2005), Salsola kali L. (Gardea-Torresdey et al., 2005),Typha angustifolia L. (Dong et al., 2007), Ipomoea aquatica Forssk. (Weerasinghe et al.,2008).

Wetlands plants have been shown to be useful for removal of Cr (Xu and Jaffe, 2006).Scirpus americanus Pers. is a common wetland plant, which occurs naturally on the pond“San German.” Recently, artificial wetlands using S. americanus Pers. have been usedto treat metal-contaminated effluent (Pacheco-Aguilar et al., 2008). In the rhizosphere ofhyperaccumulating plants, the microbial populations in general were larger than in bulksoil and they showed higher ratios of metal resistant bacteria (Aboudrar et al., 2007).

Heavy metals, such as chromium (VI) cannot be destroyed but their oxidation statemay be transformed, leading to a consequent variation in their toxicity. In fact, differ-ent aerobic and anaerobic microorganisms are capable of reducing Cr(VI). The aerobicmicrobial reduction of Cr(VI) is commonly catalyzed by soluble enzymes in many bac-teria including Pseudomonas ambigua G-1, Pseudomonas putida MK1, Escherichia coli,(Suzuki et al., 1992; Ackerley et al., 2004) and by membrane-associated reductases re-cently isolated from Bacillus megaterium TKW3 (Cheung et al., 2006). Under anaerobicconditions, both soluble and membrane-associated enzymes of the electron transfer systemcatalyzing the electron shuttle along the respiratory chain. In this process, the chromiumserves as terminal electron acceptor. (Lovley and Phillips, 1994; Wang and Shen, 1995;Tebo and Obraztsova, 1998). The reduction of Cr(VI) to Cr(III) is an effective method ofdetoxification of Cr(VI)-polluted waters. Thus, at least in some environments, the bacterialreduction of toxic ion Cr(VI) offers a potentially cost-effective bioremediation approach.Nothing has been published on the ability of rhizospheric communities of Scirpus amer-icanus Pers. to alter Cr(VI) levels in situ. Therefore, the objective of the present studywas to determine the metal’s bioaccumulation in S. americanus Pers. and to characterize


REDUCTION OF CR(VI) BY RHIZOSPHERIC BACTERIA 319

the Cr(VI) resistance and reduction capacity of rhizospheric bacterial strains as efficientcandidates in biotransformation processes.

MATERIALS AND METHODS

Sampling

Sediment and plant samples were collected during 2005, from San German pondGuanajuato, Mexico. During the summer and winter, the samples were collected from threerandom sites. Sediment samples of 1 kg were taken at sites distant at least 50 cm from plants.Samples of rhizosphere consisted of soil adhering to the roots. All samples were labeledand placed separately in polyethylene bags. A minimum of nine mature individual samplesof S. americanus Pers. plants were also collected at each site. Samples were analyzed byglass electrode or pH paper for temperature and pH immediately after collection and storedat 4◦C before analysis.

Heavy Metal Analysis in Plants and Sediment

For the determination of metals and metalloids (As, Cd, Cr, Se) in the root and aerialparts, the plants were rinsed three times with deionized water to remove contaminationfrom the soil. The plants and sediment samples were dried at 70◦C until the weight wasconstant. Samples of 0.5 g were taken and transferred to a 100 ml boiling tube along with 5ml of concentrated nitric acid. The samples were placed onto a digestion system block (40spaces) (Tecator 1016, Tecator. AB, Sweden) and then refluxed at 130◦C for four hours.The digests were filtered into separate volumetric flasks, diluted with deionized water, andeach made up to a volume of 50 ml with mixing. The metal concentrations were measuredwith ICP-AES (Inductively coupled plasma-atomic emission spectrometry, IRIS IntrepidII Model XDL, Thermo Electron Corporation, Franklin, Mass., USA) according to EPAMethod 6010.

Microbiological Analysis

Chromium tolerant bacteria isolation. Bacteria tolerant to Cr(VI) were iso-lated from rhizosphere and sediment samples using the plate count technique (Brimet al., 1999). For the isolation and culturing of strains, two modified basal mineral mediawere used containing: solution A, 100 mL L−1 (in g L−1): CaCl2.2H2O, 0.793; NaCl, 0.08;KNO3, 1.03; NaNO3, 6.98; MgSO4.7H2O, 1.00; nitrilotriacetic acid, 1.00; solution B, 10 mlL−1 (in mg L−1): FeSO4.7H2O, 200.00; ZnSO4.7H2O, 10.00; MnSO4.H2O, 3.00; H3BO3,30.00; CoSO4.7H2O, 24.00; CuSO4.5H2O, 1.00; NiCl2.6H2O, 1.70; NaMoO4.2H2O, 3.00;EDTA, 500.00); NH4SO4, 0.50 g L−1; sodium gluconate, 10 mmol L−1 and Cr(VI) 0.2mmol L−1, pH 7.0 (Francisco et al., 2002). A filter-sterilized solution of K2CrO4 was usedas the source of Cr(VI). The basal mineral medium without Cr(VI) was used for estimatingtotal bacterial population. Plates were incubated at 28◦C for 3–5 d, and the total numberof bacterial isolates was determined as colony-forming units per gram (cfu g−1). Thirteenmorphologically distinct colonies attained from rhizosphere samples were selected as Cr-resistant isolates. These isolates were purified and stored at −70◦C in Luria-Bertani (LB)broth medium containing 50% glycerol and 0.2 mM Cr(VI) until further use.



Chromium resistance. The thirteen rhizospheric isolates resistant to 0.2 mMCr(VI) were used in the following assays. The bacteria were grown overnight in 250 mLErlenmeyer flasks containing 100 ml of Luria-Bertani (LB) medium in a rotary shakerat 150 rpm at 28 ◦C until the population reached around 109 cells mL−1. Bacterial cellswere harvested by centrifugation (14000 g for 10 min at 4◦C) followed by washing thecell pellet in phosphate buffer (0.1 M KH2PO4-K2HPO4; pH, 7.0). After two washes inphosphate buffer, the cells were resuspended in the same buffer. Chromate removal capac-ity was evaluated by resting cell suspensions. Aliquots of 20 ml (OD600 nm equivalent toapproximately 3 × 108 cell mL−1) from each of these bacterial suspensions were supple-mented with Cr(VI) to a final concentration of 20 µM. Lysed cells were used as negativecontrols for the Cr(VI) reduction activity (Sultan and Hasnain, 2007). They were incubatedin a rotary shaker (125 rpm) at 28◦C for 1 hour. Residual chromate was measured by the1,5-diphenylcarbazide method (DFC) after removing the cells by centrifugation (Rodier,1981). Tolerance (Minimum Inhibitory Concentration [MIC]) to chromium was examinedby following the turbidity (OD600 nm) of cultures supplemented with K2CrO4 to obtainchromate (Cr(VI)) at final concentrations of 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5 and 5mM in Tris minimal medium (Mergeay et al., 1985). Tubes containing 5 ml of Tris minimalmedium were inoculated with 1.5 × 106 cells and incubated aerobically in a rotary shaker(150 rpm) for 3–5 d at 28◦C. The growth of bacteria was monitored by turbidity (OD600nm). The MIC was measured as the lowest concentration of chromium at which there wasno growth.

Reducing capacities of isolates. The isolates capacities to reduce chromiumand nitrate under anaerobic conditions were tested in modified Tris medium (g L−1: NaCl,4.68; KCl, 1.49; NH4Cl, 1.07; NaH2PO4H2O, 0.5147; MgCl26H2O, 0.20; CaCl22H2O,0.03); sodium gluconate (10 mM); Sol. B 1 ml L−1 (pH 7.0) and one of the followingcompounds: 100 µM Cr(VI) or 0.05% KNO3. The tests were performed using vacutainertubes (10 mL) injected with 6 mL of basal culture medium previously deoxygenated inan anaerobic box by bubbling with chromatographic grade N2. The remaining chromiumwas determined as outlined above and nitrate respiration was assayed by measuring thepresence of gas, nitrate and nitrite (Anderson and Ingram, 1993).

To test sulfate reduction, vacutainer tubes (10 mL) with modified saline Postgate’smedium, containing (g L−1): sodium lactate, 4.00; yeast extract, 1.00; sodium citrate,0.30; K2HPO4, 0.50; MgSO4, 2.00; NH4Cl, 1.00; Fe(NH4)(SO4)2.6H20, 0.20; NaCl, 7.00(pH 7.2), were inoculated with the bacterial isolates (Telang et al., 1999). Ferric-reducingcapacity assays were performed using vacutainer tubes (10 mL) with V10 culture medium(Valencia-Cantero et al., 2003) amended with ferric citrate at 250 µM final concentration,inoculated to 1.5 × 106 cells mL−1 and incubated at 28◦C for 15 d. The concentrationof Fe2+ in the cultures was determined by the ferrozine assay as previously described(Sørensen, 1982). Dissolved sulfide content was measured by the methylene blue method(Cline, 1969).

Characterization of chromium reduction. The isolates were preculturedovernight in 100 ml Erlenmeyer flasks containing 25 mL of LB medium. The cellswere harvested by centrifugation, washed, and resuspended in phosphate buffer (0.1 MKH2PO4-K2HPO4 pH 7.0). Chromate removal capacity of the strains was evaluated atdifferent initial pH (7.0–9.0) and different NaCl concentrations (0.5–1.5 M) in modifiedTris basal media supplemented with 0.2 mM Cr(VI) (pH 7.0). The basal medium wasinoculated with 1.5 × 106 cells mL−1 bacterial suspension and incubated aerobically at28◦C on a rotary shaker (150 rpm) for 48 h. The concentration of remaining chromium



was determined at the end of the experiment by the 1,5-diphenylcarbazide method (DFC)(Rodier, 1981). Chromate-reducing activity expressed as Cr(VI) removal percentage wasestimated as the concentration difference Cr(VI) between inoculated cultures and sterilecontrols. Bacterial growth was monitored at the end of the experiment by measuring opticaldensity of the cultures at 600 nm and cells counting using a Petroff Hausser countingchamber. All the experiments were done in triplicate.

Cr(VI) reduction by mixed cultures. Bacterial strains S17 and S28 were precul-tured separately in flasks each containing 100 ml of LB medium with 0.2 mM Cr(VI) forstimulation. Cultures were incubated aerobically at 28◦C with constant shaking at 150 rpmuntil the cultures reached an optical density of 0.54 (S17 strain) and 0.80 (S28 strain) at600 nm. The cells were then washed and reinoculated (2 × 106 cells mL−1) in mono andmixed cultures with Tris-modified (minimal salts) medium supplemented with 0.2 mMCr(VI). Simultaneously, Cr(VI) free media inoculated either with mono-culture or withmixed-culture as well as uninoculated media containing Cr(VI) served as controls. All theflasks were incubated for 48 h at 28◦C in a rotary shaker at 150 rpm. Three replicates fortreatment were tested.

Bacterial growth was monitored at time intervals by measuring optical density of thecultures at 600 nm and by plate count techniques. Each strain presents a distinct colonialmorphology allowing the enumeration of each one of them in the same plates. Hexavalentchromium in the supernatants was measured by the 1,5-diphenylcarbazide method (DFC)(Rodier, 1981). Total chromium concentration in the supernatants and cellular pellets wasmeasured with FAAS (Flame Atomic Absorption Spectrometry, TJA Unicam Model 969Solar AA). The samples were digested previous to the FAAS measurements with HNO3

for 30 min at 160◦C.Cr(VI) reduction in a sample of the industrial effluent. Effluent samples

from pond San German were collected in sterilized bottles and the heavy metal con-centrations were measured with ICP-AES. Cr(VI) in the effluent was measured by the1,5-diphenylcarbazide (DPC) method (Rodier, 1981).

Isolates S17 and S28 were precultured as described above. Erlenmeyer flasks con-taining 30 ml of sterile or non-sterile effluent of San German pond supplemented with 10%of Tris modified medium were inoculated with 2 × 106 cells mL−1 in mono and mixed cul-tures. The effluent had been previously sterilized by filtration through 0.22 µm pore-size,membrane filters (MF-Millipore). The cultures were incubated for 48 h at 28◦C in a rotaryshaker at 150 rpm. The amount of reduced Cr(VI) and bacterial growth (OD600 nm) weremeasured as previously described at the end of the experiment.

rRNA Gene Analysis

PCR amplification of the 16S ribosomal RNA subunit gene was performed asdescribed by Weisburg et al. (1991) using forward-primer fD1 (5′-CCGAATTCGTCGACAACAGAGTTTGATCCTGGCTCAG- 3′) and reverse-primer rD1 (5′-CCCGGGATCCAAGCTTAAGGAGGTGATCCAGCC-3′). The amplification product was cloned into thepCR2.1 TOPO-Cloning vector (Invitrogen). Plasmids with the 16S fragment were purifiedusing the PureLinkTM Quick Plasmid Miniprep kit (Invitrogen) according to the manufac-turer’s instructions. The nucleotide sequence of the cloned fragment was determined by thedideoxy chain termination method (Sanger et al., 1977) using an automated DNA sequencer(ABI 37300×l DNA, PE Applied Biosystems, Inc.). Sequencing was achieved with primers



fD1 and rD1 described above. The resulting sequences were compared against reported se-quences of the NCBI GenBank using the BLAST program (www.ncbi.nlm.nih.gov/blast).The aligned sequences were then analyzed by the distance matrix method by using theJukes-Cantor method of the Mega 3.1 software package (Molecular Evolutionary GeneticsAnalysis). The phylogenetic tree (see Figure 1) was constructed using the Neighbor-Joiningmethod with bootstrap analysis of 1000 resamplings using Mega 3.1. Thirteen 16S rRNAgene sequences were obtained from the isolates and deposited on the NCBI GenBank withaccession numbers from EU747692 to EU747704.

Statistics

All data represent the mean of three replicates. Standard deviation is indicated wher-ever appropriate. ANOVA was conducted for all data and Tukey’s test was used for testingthe difference between the treatments by using the SAS statistical software package (Sta-tistical Analysis System).

RESULTS

The analysis of sediment from San German pond showed the presence of heavymetals: Cr, As, Se, Cd at concentrations of 409, 8, 3, 3 mg kg−1 dry wt respectively(Table 1). Cr showed the highest concentration in the sediment with a significant difference(p ≤ 0.05) when compared to the other metals again. Scirpus americanus Pers. extracts andaccumulates these metals in some of its tissues. Metal concentrations in the plant showedthat Cr, As, Cd, Se in the aerial tissues were maintained at low levels (Cr: 1, As: 9, Cd: 23and Se: 5 mg kg−1 dry wt). In contrast, the metals in the plant root system showed highconcentrations (Cr: 970, As: 49, Cd: 41 and Se: 85 mg kg−1 d. wt). The bioaccumulationfactor of these metals in the plant showed that As and Cr presented significant differences(p ≤ 0.05) with regard to Cd and Se. Interestingly, only the roots concentrate significantlevels of Cr.

Table 1 Metal bioconcentration in roots and aerial tissues of Scirpus americanus Pers

Metal

Treatment As Cr Cd Se

mg kg−1 dry weightSediment 82 b3 409 b 3 c 3 bRoot 49 a 970 a 41 a 85 aAerial tissues 9 b 1 c 23 b 5 bWhole plant 58 971∗ 65 90Bioaccumulation factor1 7 ± 3.6 2 ± 0.1 19 ± 0.1 24 ± 2.9

1Bioaccumulation factor is metal concentration in plant at harvest/concentration of metal in sediment.2Each value represents the average of three replicates ± standard deviation.3,∗Different letters within a column indicate significant statistical difference by Tukey’s test (p ≤ 0.05).



S18

Microbacterium hydrocarbonoxydans DSM...

Microbacterium sp. XT11 (DQ350882)

S19

Microbacterium sp. VKM Ac-2050 (AB042...

Microbacterium sp. VKM Ac-2015 (AB042...

Curtobacterium flaccumfaciens pv. fla...

S20

Curtobacterium flaccumfaciens pv. fla(2)

S14

Arthrobacter sp. GOL01 (AY940423)

Arthrobacter nicotianae SB42 (AJ315492)

S15

Arthrobacter sp. BWDY-45 (DQ314547)

Bacterium W8 (AY345527)

S11

Arthrobacter sp. P2 (DQ288888)

Rhodococcus sp. HS1(AY168579)

Rhodococcus sp. HS6 (AY168584)

S23

Agrobacterium tumefaciens (DQ468100)

Agrobacterium tumefaciens 204 (EF217305)

S17

S21

Xanthomonas retroflexus (AY841369)

Stenotrophomonas maltophilia e-a21 (A...

S28

Pseudomonas aeruginosa PAL106 (DQ464061)

Pseudomonas sp. OLB-1 (AJ387904)

Pseudomonas sp. HR 13 (AY032725)

Uncultured bacterium clone Lan-75 (AY...

S27

Uncultured Pseudomonas sp. DS096 (DQ2...

Pseudomonas sp. K3zhy (AM410627)

Pseudomonas sp. 28zhy (AM410623)

Pseudomonas fluorescens F (DQ146946)

S29

Pseudomonas synxantha (AF267911)

S26

100

97

100

80

98

100

96

93

100

100

43

100

60

100

100

83

100

44

83

99

100

96

96

83

82

91

92

71

0.02

Figure 1 Phylogenetic tree of the partial sequences of the 16S rRNA gene of the rhizospheric bacterial isolatesobtained using neighbor-joining analysis. Bootstrap probabilities are shown in percentages of 1000 replicates.Scale bar, 0.02 divergent residues per site.



Bacterial Isolation and Characterization

The viable bacterial populations determined in chromium free media were 1.6 × 109

cfu g−1 for rhizosphere and 4.5 × 108 cfu g−1 sediment. However, the rhizospheric bacterialpopulation evaluated in the presence of 0.2 mM Cr(VI) was 2.3 × 108 cfu g−1 soil whereasfor sediment was 1.6 × 107 cfu g−1 sediment.

Thirteen bacterial isolates were obtained from rhizosphere. Based on the phylogenyof the 16S rRNA gene characterization, the isolates showed similarities to previously re-ported species and were comprised of actinobacteria, and alpha and gamma-proteobacterialsubgroups. The strains S18 and S19 were related to Microbacterium cluster (99%). S11,S14, and S15 showed high similarity (99%) to Arthrobacter, whereas strain S20 was closelyrelated to Curtobacterium and S23 to Rhodococcus. These genera fall within the Actinobac-teria. The group of alpha-proteobacteria was represented by strain S17 and was similar toAgrobacterium. The gamma-proteobacteria group conformed to a Pseudomonas cluster(S26, S27, S28, and S29) and a Xanthomonas cluster (S21). All these microorganisms wereprevalent in this system (Figure 1).

The intrinsic tolerance of the strains to potassium chromates was measured. MIC(Mimimum Inhibitory Concentration) for Cr(VI) varied among the strains. Curtobacteriumsp. strain S20 and Rhodococcus sp. strain S23 showed Minimum Inhibitory Concentrationto Cr(VI) of 5.0 mM (Table 2). Six of the isolates; Arthrobacter sp. strain S11, Arthrobactersp. strain S14, Arthrobacter sp. strain S15, Microbacterium sp. strain S18, Microbacteriumsp. strain S19 and Pseudomonas sp. strain S29 were tolerant to Cr with MICs of 4.0–4.5mM. All of the Cr(VI)-resistant isolates could reduce chromate, and the strains S11, S14,S15 and S29 showed the highest removal capacity and reduced more than 40% of Cr(VI)(20 µM) in the resting cell test within 1 h.

Table 2 Cr(VI) tolerance and removal by bacterial isolates

Cr(VI) removal%2

MIC1

Isolate [mM] Cr(VI) Resting cell3 Dead cell4

S11 4.5 42.8 ± 0.725 0.1 ± 0.007S14 4.5 40.5 ± 0.35 10.5 ± 0.21S15 4.0 41.9 ± 0.04 0.1 ± 0.02S18 4.0 7.5 ± 0.14 0.1 ± 0.004S19 4.0 5.1 ± 0.14 0.1 ± 0.003S20 5.0 9.3 ± 0.22 11.8 ± 0.21S23 5.0 26.6 ± 0.42 2.5 ± 0.07S17 0.5 19.8 ± 0.21 0.1 ± 0.002S21 2.0 34.7 ± 0.07 0.1 ± 0.003S26 3.0 20.7 ± 0.20 0.1 ± 0.006S27 2.0 12.3 ± 0.14 5.2 ± 0.03S28 1.5 23.4 ± 0.21 5.0 ± 0.01S29 4.0 42.4 ± 0.29 2.1 ± 0.14

1Cr(VI) Minimum Inhibitory Concentration for isolates incubated in Tris minimal medium for 3–5 d at 28◦C (seeMethods).2Cr(VI) removal percentage is the difference between inoculated, live or dead cultures and sterile controls; eachvalue represents the average of three replicates ± standard deviation.3,4Cultures were incubated in phosphate buffer supplemented with 20 µM Cr(VI) at 28◦C for 1 h under aerobicconditions (see Methods).



Table 3 Cr(VI) removal at different pH values and salinity by bacterial isolates

pH1 NaCl

Strain 7 8 9 0.5 M 1.0 M 1.5 M

Cr(VI) removal%2

S11 29 a3 24 b 22 b 24 0.0 0.0S17 53 a 44 b 16 c 8 a 4 b 0.0S26 44 a 35 b 11 c 10 a 4 b 3 bS27 61 a 56 a 42 b 6 a 1 b 0.0S28 99 a 98 a 93 b 11 a 2 b 0.0Control 4 4 4 4 4 4

1Cultures were incubated aerobically in modified Tris basal media supplemented with 0.2 mM Cr(VI) at 28◦C for48 h (see Methods).2Cr(VI) removal percentage is the difference between inoculated cultures and sterile controls.3Each value represents the average of three replicates; different letters within a row indicate significant statisticaldifference by Tukey’s test (p ≤ 0.05).

Strains S11, S14, S15, S18, S19, S20, and S23 are Gram-positive. Strains S17, S21,S26, S27, S28 and S29 correspond to Gram-negative bacteria. Some of them, such as S23,S17, S26, S27, and S28 reduce sulfate, and S14, S15, S20, S23, S17, S21, and S27 reducenitrate when oxygen is limiting. Strain S28 is a denitrifying bacterium, and all strainsreduced chromate and iron under anaerobic conditions.

Effect of Different pH Values and NaCl Concentration in Chromate

Reduction in Culture Media

The initial pH of the culture medium was shown to be an influential factor for growthand Cr(VI) reduction. The strains S11, S17, S26, S27 and S28 could grow and reduceCr(VI) in a wide range of pH (7–9) (Tables 3 and 4). However, the maximum growth, aswell as Cr(VI) reduction by the strains tested was observed at pH 7 with 29, 53, 44, 61, and99% Cr(VI) reduction (at 0.2 mM Cr(VI) initial concentration) respectively. In regard to

Table 4 Bacterial growth at different pH values and salinity in the media supplemented with 0.2 mM Cr(VI)

pH1 NaCl

Strain 7 8 9 0.5 M 1.0 M 1.5 M

106 cell mL−1

S11 371 a2 292 b 273 b 309 a 26 b 10 bS17 45 a 36 b 8 c N.d.3 N.d. N.d.S26 338 a 275 b 267 b 50 a 4 b 4 bS27 84 a 80 a 72 b N.d. N.d. N.d.S28 46 a 44 a 34 b 5 N.d. N.d.

1The treatments were inoculated with 7.5 × 104 cell mL−1 then were incubated aerobically in modified Tris basalmedia at 28◦C for 48 h (see Methods).2Each value represents the average of three replicates; different letters within a row indicate significant statisticaldifference by Tukey’s test (p ≤ 0.05).3Not detected.



Figure 2 Cell reduction of Cr(VI) over 48 h. Agrobacterium sp. strain S17 and Pseudomonas sp. strain S28 weregrown in mixed culture at 28◦C, and 0.2 mM Cr(VI). S17-S28 reduction of Cr(VI) (×). Cell growth of S17 with0.2 mM Cr(VI) (�). Cell growth of S28 with 0.2 mM Cr(VI) (•). Cell growth of S17 in control (�). Cell growthof S28 in control (◦). Results are the average of triplicate experiments.

the effect of NaCl on the Cr(VI) reduction by the same strains, the maximum cell growthand chromate reduction occurred at 0.5 M NaCl, and corresponded to Cr(VI) reduction of24, 8, 10, 6, and 11% at 0.2 mM Cr(VI) initial concentration respectively. The presence ofNaCl reduced the rate of Cr(VI) reduction.

Development of the Bacterial Consortium and Removal of Cr(VI) in an

Industrial Effluent

A mixed inoculum (strains S17 and S28) grown in aerobic conditions showed asignificant reduction of chromium during the log phase (Figure 2). The chromium wastotally reduced within an incubation time of 12–30 h. The specific growth rates were 4.3and 2.9 h−1 for strains S17 and S28, respectively. These growth rates were calculatedin the exponential phase according to Madigan et al. (2000). The mixed culture withoutchromium had specific growth rates of 3.6 and 2.8 h−1 (Figure 2). The growth rates forS17 and S28 in mono cultures were 5.2 and 3.8 h−1, and they reduced 96 and 72% of thechromium respectively in 48 h at initial Cr(VI) concentration of 0.2 mM. In cultures withoutchromium, the specific growth rates for both were 4 h−1 (Figures 3 and 4). The reductionof chromium by mixed culture (strains S17 and S28) occurred mainly from 12 to 30 h ofincubation. In general terms, Cr(VI) reduction under aerobic conditions occurred duringthe log phase. No change in Cr(VI) concentration was detected in control treatments.

The analysis of the industrial effluent indicated the presence of 81 µg L−1 of Cr(VI)and other heavy metals (Se, Pb, Cr, Cd, and As at 1.8, 1.2, 1.1, 0.9, and 0.6 mg L−1

respectively). After an incubation of 48 h in the presence of the consortium Cr(VI) wasreduced 64.5 and 64.1% of 81 µg L−1 in sterile effluent and non-sterile effluent respectively(Table 5). The rate of reduction of chromium by the consortium increased in the assaysupplemented with 10% of medium under either condition (93.8 and 69.0% respectively of81 µg L−1 Cr(VI) initial concentration).



Figure 3 Cell reduction of Cr(VI) over 48 h. Agrobacterium sp. strain S17 was grown in monoculture at 28◦C,and 0.2 mM Cr(VI). S17 reduction of Cr(VI) (×). Cell growth of S17 with 0.2 mM Cr(VI) (�). Cell growth ofS17 in control (�). Results are the average of triplicate experiments.

DISCUSSION

The pond of San German, Guanajuato Mexico, is highly contaminated by differentindustrial sources, particularly by a chromate factory and leather tanning (Armienta andRodrıguez, 1995). Sediment samples showed a great variation in metal content (Cr, As, Cd,Se), Cr was the predominant metal (409 mg Cr kg−1) at the time of our study. S. americanusPers. showed the capacity to extract Cr, As, Cd, Se from its aquatic surroundings. All themetals were accumulated in the root system. In particular, chromium concentration in theroots was 970 times higher than that of the aerial tissues (Table 1), indicating a limitedtranslocation of Cr to the aerial tissues of the plant. Similar behavior has been shown for

Figure 4 Cell reduction of Cr(VI) over 48 h. Pseudomonas sp. strain S28 was grown in monoculture at 28◦C,and 0.2 mM Cr(VI). S28 reduction of Cr(VI) (×). Cell growth of S28 with 0.2 mM Cr(VI) (•). Cell growth ofS28 in control (◦). Results are the average of triplicate experiments.



Table 5 Chromium (VI) removal and growth of the bacterial consortium (S17 and S28) in an industrial effluent

Cell growth (OD600 nm)1

Effluent Consortium S17 S28 Uninoculated

Sterile effluent 0.152 0.14 0.06 0.02Sterile effluent supplemented with culture medium 0.28 0.22 0.14 0.02Non-sterile 0.12 0.09 0.04 0.02Non-sterile supplemented with culture medium 0.13 0.13 0.06 0.03

Cr(VI) removal%Sterile effluent 64.5 24.6 0.3 0.0Sterile effluent supplemented with culture medium 93.8 40.3 18.1 0.0Non-sterile 64.1 23.2 0.3 0.2Non-sterile supplemented with culture medium 69.0 34.9 10.1 1.0

1Cultures were incubated aerobically at 28◦C for 48 h and Cr(VI) removal was measured at the end of theexperiment.2Each value represents the average of three replicates.

other wetland plants (Zurayk et al., 2001). This limited Cr translocation could be a result ofthe reduction process of Cr(VI) to Cr(III) in plant roots (Lytle et al., 1998) and the tendencyof the latter to be immobilized in the vacuoles of root cells, being less toxic for the plant(Shanker et al., 2004). The strategy of S. americanus Pers. as a pseudometallophyte plantis a significant metal immobilization by the roots, thereby making this plant a potentialcandidate for phytostabilization.

The size of the rhizospheric bacterial population tolerant to Cr(VI) was 14.3 timeslarger than the population away from the rhizosphere, possibly the specific rhizosphereconditions could reflects a greater tolerance of bacteria to Cr. This observation was similarto that reported for Thlaspi caerulescens J.Presl & C.Presl grown in Zn contaminated soils(Delorme et al., 2001). The high concentration of bacteria around the roots (rhizosphere)occurs by the production of root exudates that can be used to support bacterial growth(Penrose and Glick, 2001). Bacterial inoculation in the root zone for metals uptake inScirpus americanus Pers. is unknown, but it has been published that some microorganismsmay excrete organic compounds which increase bioavailability and facilitate root absorptionof essential metals, such as Fe (Crowley et al., 1991) and Mn (Barber and Lee, 1974) as wellas nonessential metals, such as Cd, Ni, Zn and Cr (Salt et al., 1995; Whiting et al., 2001;Abou-Shanab et al., 2003; Faisal and Hasnain, 2005a). Also rhizospheric microorganismscan directly influence metal solubility by altering the metal’s oxidation state (Faisal andHasnain, 2005b).

Several reports have been made on different strains that can reduce the solubleand toxic Cr(VI) to nontoxic and immobile Cr(III) under aerobic conditions (Faisal andHasnain, 2004). In the present study the rhizospheric isolates were able to reduce Cr withoutabsorbing the metal based on the assays of resting cells. Considerable reduction of Cr(VI)has been observed at an initial concentration of 20 µM; this is very similar for other bacteria(Branco et al., 2004).

The Cr(VI)-tolerant strains reported in this study as Curtobacterium sp. strain S20 andRhodococcus sp. strain S23 could grow at 5 mM Cr(VI) in minimal medium (Table 2). Faisalet al. (2005) observed that strains (Ochrobactrum intermedium CrT-1 and Bacillus cereus S-6) isolated from tannery effluents could grow at 52 mM Cr(VI) in acetate-minimal medium.



Most Cr(VI)-resistant microorganisms isolated from rhizosphere of Alyssum murale Waldst.& Kit. as genus Arthrobacter could grow at 5 mM Cr(VI), species of Microbacteriumtolerate from 1 to 5 mM Cr(VI) and Pseudomonas riboflavina could grow at 0.5 mM inminimal medium (Abou-Shanab et al., 2007). In our study, these genera presented similarvalues with the exception of genus Pseudomonas that grew from 1.5 to 4 mM Cr(VI) inminimal medium. Polti et al. (2007) reported different actinomycete strains isolated fromwaste water of a copper filter plant and sugar cane plant, as Streptomyces can tolerate from5 to 17 mM Cr(VI).

In the present study the strains were identified phylogenetically on a partial 16SrRNA sequence. Analysis of the isolates showed that the rhizosphere of S. americanusPers. presents different bacterial populations, some isolates correspond to the genera Mi-crobacterium, Arthrobacter and Pseudomonas which have been reported in the rhizosphereof Ni hyperaccumulating plants (Abou-Shanab et al., 2006; Abou-Shanab et al., 2007), andsediments, tannery waste and tannery waste contaminated soil with capacities of resistanceand reduction Cr(VI) (DeLeo and Ehrlich, 1994; Pattanapipitpaisal et al., 2001; Asatianiet al., 2004; Rajkumar et al., 2005). Similarly, Curtobacterium and Rhodococcus havebeen isolated from the rhizosphere of Thlaspi goesingense Halacsy showing the capacity ofresistance to Ni and siderophore production (Idris et al., 2004). Strains of Rhodococcus ery-thropolis MtCC 7905 have been isolated from metal contaminated soil, the strains reducedCr(VI) and showed plant growth promotion (Trivedi et al., 2007). The genus Agrobacteriumhas also been isolated from contaminated soil with chromate reduction capacity (Lloveraet al., 1993). Strains of Xanthomonas have been isolated from hypersaline environments ofwater and sediment samples resistant to chromium (Rıos et al., 1998) (Figure 1).

The capacity to reduce nitrate, sulfate and iron, and to grow at different pH valuesand NaCl concentration varied among the isolates. The strains corresponding to Gram-positive bacteria can reduce nitrate (Arthrobacter sp. S14 and S15; Curtobacterium sp.S20; Rhodococcus sp. S23). Only strain S23 reduced sulfate. The pseudomonads wereheterogeneous in their reduction of different electron acceptors. Strains S17, S27, and S28reduced nitrate and sulfate. Strain S28 is a denitrifier. All of these strains reduced chromiumunder anaerobic conditions. As suggested by Holmer and Storkholm (2001) the anaerobicrespiration that occurs in lakes sediments among the different strains could have beendriven by the low oxygen tension in sediments and the mineralization of organic mattermay involve alternative electron acceptors such as Fe(III), nitrate, or sulfate.

In the present study different pH intervals and NaCl concentrations were evaluated onthe Cr(VI) reduction. These parameters change throughout the year in the pond under studydue to seasonal conditions. The strains belonging to the genus Pseudomonas (S27 and S28)showed the highest percentage of chromium removal, followed by an Agrobacterium isolate(strain S17) and Arthrobacter (strain S11) (Table 3). The results clearly showed a directnegative correlation between Cr(VI) reduction and pH within the range of 7–9. Growthwas inhibited at high pH (above 7), and Cr(VI) reduction was suppressed as well (Table 4).Because chemical compounds with Cr(VI) are soluble over a wide pH range and generallymobile in soil–water systems (Kamaludeen et al., 2003), this loss of Cr(VI) reductionindicates that cell growth is a prerequisite for enhanced reduction to occur. According toElangovan et al. (2006), reduction of chromium by a Bacillus sp. strain occurred at pH5.5 to 8 and was strongly inhibited at pH 4 to 5, while optimum pH for complete Cr(VI)reduction by an Escherichia coli ATCC 33456 was 6.5 (Bae et al., 2005). Since Cr(VI)reduction is enzyme-mediated, pH changes affect the enzymatic reactions (Lee et al.,2008).



In the present study, the salinity of the growth medium also had a great impact onCr(VI) removal by the strains studied and their growth. In the absence of added salts,chromium removal and bacterial growth were detected. The results clearly indicated thatstrains (S11, S26, and S28) were halotolerant (they grew at 0.5 M NaCl see Table 4), asdefined by Horikoshi (1999). The effects of different concentrations of NaCl on removalof chromate (0.2 mM) indicated that only Pseudomonas sp. strain S26 removed chromiumat 1.5 M NaCl (Table 3). At higher NaCl levels, the low reduction rates observed may bedue to the toxicity, which would decrease the number of viable cells.

Agrobacterium sp. strain S17 and Pseudomonas sp. strain S28 were studied forCr(VI) reduction under aerobic conditions in mixed culture. The growth profile of thebacterial consortium (S17 and S28) was significantly higher than either monoculture withchromium present. At an initial Cr(VI) concentration of 0.2 mM, a complete reduction ofCr(VI) occurred within 30 h for the consortium while the monocultures (strains S17 andS28) showed a reduction of only 89.6 and 62.7% respectively (Figs 2, 3, 4). Therefore,the reduction capacity of Cr(VI) by mixed cultures as described by Alexander (1971) forbacterial interactions seemed to be a cooperative action, which in the present study canbe explained in terms of a larger population, since these strains seem to participate inrelationships entailing the simultaneous cooperation (protocooperation) by reducing theirgeneration time when growing together. We did not detect chromium in cellular pellets(data not shown). However, a slower increase in cell growth occurred in the mono andmixed culture with chromium than in cultures without it. This variation could result fromstress induced by chromium on the bacteria.

When the bacterial consortium was tested in the industrial effluent Cr(VI) was re-duced. The reduction rate by the consortium (strains S17 and S28) was higher than eithermonoculture. This consortium could provide a higher level of tolerance to metal ions (Se,Pb, Cd, and As) present in the effluent than individual strains. Similar observations havebeen made in the case of cocultures of Escherichia coli ATCC 33456 and Pseudomonasputida DMP-1 by Shen and Wang (1995). In the present study, the reduction rate in-creased when the effluent (sterile and non-sterile) was supplemented with culture medium(Table 5). The effluent could have been deficient in certain nutrients, which limited thegrowth of Cr(VI)-reducing bacteria in leather tanning and electroplating effluents inoc-ulated with Pseudomonas aeruginosa strain A2Chr (Ganguli and Tripathi, 1999). Thedevelopment of this bacterial consortium (S17 and S28), which was able to tolerate andefficiently transform chromium to a less toxic form, may be a promising way for bioreme-diation of contaminated wastes.

ACKNOWLEDGMENTS

This work was supported by National Council of Science and Technology (Conacyt).We thank Dr. Gene Bartholomew for valuable help in reviewing the manuscript. We extendour thanks to the anonymous reviewers for their positive and relevant comments thatimproved the manuscript.

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Isolation and Characterization of Hexavalent Chromium-Reducing Rhizospheric Bacteria From a Wetland

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