research article resistance to and accumulation of heavy

Research ArticleResistance to and Accumulation of Heavy Metals byActinobacteria Isolated from Abandoned Mining Areas

Soraia El Baz1 Mohamed Baz1 Mustapha Barakate1 Lahcen Hassani1

Abdelhay El Gharmali2 and Boujamacirca Imziln1

1Environmental Microbiology and Toxicology Unit Laboratory of Biology and Biotechnology of Microorganisms (LBBM)Faculty of Sciences Semlalia Cadi Ayyad University PO Box 2390 40000 Marrakech Morocco2Laboratory of Hydrobiology Ecotoxicology and Assainissement (LHEA) Faculty of Sciences Semlalia Cadi Ayyad UniversityPO Box 2390 40000 Marrakech Morocco

Correspondence should be addressed to Boujamaa Imziln imzilnucama

Received 7 August 2014 Revised 13 November 2014 Accepted 18 November 2014

Academic Editor Wen-Jun Li

Copyright copy 2015 Soraia El Baz et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Accumulation of high concentrations of heavy metals in environments can cause many human health risks and serious ecologicalproblems Nowadays bioremediation using microorganisms is receiving much attention due to their good performance The aimof this work is to investigate heavy metals resistance and bioaccumulation potential of actinobacteria strains isolated from someabandonedmining areas Analysis of mining residues revealed that high concentration of zinc ldquoZnrdquo was recorded in Sidi BouatmanArbar and Bir Nhass mining residuesThe highest concentration of lead ldquoPbrdquo was found in Sidi Bouatman Copper ldquoCurdquo cadmiumldquoCdrdquo and chromium ldquoCrrdquo were found with moderate and low concentrationsThe resistance of 59 isolated actinobacteria to the fiveheavy metals was also determined Using molecular identification 16S rRNA these 27 isolates were found to belong to Streptomycesand Amycolatopsis genera The results showed different levels of heavy metal resistance the minimum inhibitory concentration(MIC) recorded was 055 for Pb 015 for Cr and 010mgsdotmLminus1 for both Zn and Cu Chemical precipitation assay of heavy metalsusing hydrogen sulfide technic (H2S) revealed that only 27 isolates have a strong ability to accumulate Pb (up to 600mg of Pb perg of biomass for Streptomyces sp BN3)

1 Introduction

Heavy metal pollution is one the most important environ-mental problems in marine terrestrial and freshwater areas[1] They can be accumulated in the human body throughfoodwebs thereby promoting chronic and acute disorder andcreating obviously serious health problems [2 3]The anthro-pogenic sources of metal contamination can be divided intofive main groups (i) metalliferous mining and smelting (ii)industries (iii) atmospheric deposition (iv) agriculture and(v) waste disposal [4 5] Industries bearing heavy metalssuch as Pb Zn Cr Cd Cu As and Ni are a major concernbecause they are well known to produce a long-term negativeimpact on soil and water in the surrounding environment[6 7]

Conventional technologies for removing metals fromindustrial effluents such as chemical precipitation chemical

oxidation or reduction ion exchange membrane filtrationelectrochemical treatment reverse osmosis and evaporationwere frequently used These processes may be ineffective orextremely expensive [8] Nowadays new processes basedon microorganism technology like bioaccumulation andbiosorption have been investigated for the developmentsof cheaper and more effective technologies in order todecrease the amount of industrial wastewater produced andto improve the quality of the treated effluent Awide variety ofmicroorganisms for example bacteria yeast algae protozoaand fungi have developed the capabilities to protect them-selves from heavymetal toxicity by various mechanisms suchas uptake oxidation adsorption methylation and reduction[4] It was also reported that some bacteria can use mech-anisms of tolerance and detoxification of heavy metals andstill produce chelating agents that bound metals and reducetheir toxicity [9] Many living bacteria have been reported to

Hindawi Publishing Corporatione Scientific World JournalVolume 2015 Article ID 761834 14 pageshttpdxdoiorg1011552015761834

2 The Scientific World Journal

Studied mining siteAffluents of the Tensiftriver Road Goundafa

Haut Atlas mountain

Marrakech

Tensift river

KettaraMassif Jbilet Sidi

BouatmanBir Nhass

NN

Marrakech

Rabat

25km

500km

Figure 1 Localisation of prospected abandoned mining areas ofMarrakech Morocco Kettara Sidi bouatman Bir Nhass andGoundafa

reduce or to transform toxic contaminants into their less toxicforms Actinobacteria constitute a morphologically diversegroup of Gram-positive bacteria with high metabolic ver-satility [10] As reported earlier actinomycetes are the goodbioremediation [11ndash16] Their metabolic diversity particulargrowth characteristics mycelial form and relatively rapidability to colonize selective substrates make them well-suitedfor use as agents for bioremediation of inorganic and organiccompounds [16ndash20]

Industrial and mining activities are important for eco-nomic development especially in many developing countriesasMorocco However these activities also represent the mainsources of heavy metal contamination

InMarrakech regionmany exploitationmetalmines haveceased to be functional many years ago Mining residueswere abandoned in open air without any treatment and wereexposed to the atmospheric hazards In fact these releaseheavy metals and other pollutants to the drainage and rep-resent a potential danger of surrounding terrestrial andaquatic ecosystemsMoreover thesemining sites are generallysituated near agricultural zones [21]

To our knowledge little research had been done regardingthe problematic of mining residues in our country despiteawareness of health and environmental risks that they maycause

In the present study we are interested in mining residuescollected from various abandoned mining areas locatedaround Marrakech region West Central Morocco (Figure 1)Kettara mine (KT) is located approximately 30 km North-West of Marrakech and was operated for pyrrhotine extrac-tion until 1981 [22] Sidi Bouatman mine (SB) is locatedapproximately 33 km north of Marrakech and was operatedfor Pb Zn and pyrite extraction until 1980 The old Bir

Nhass zinc mine (BN) is located approximately 30 km northof Marrakech it was closed at the end of the 20th centuryGoundafa mine located approximately at 90 km south ofMarrakech operated for Pb Zn and Cu extraction and in1989 the mine has ceased to be functional In Goundafa areasamples were collected from two sites Arbar plant (GA) andTenfit Mine (GT)

The aim of the present work deals with the evaluation ofthe contamination levels of mining residues the isolation ofheavymetals resistant actinobacteria their potential capacityto accumulate heavy metals (Pb Cu Zn Cd and Cr)and both morphological and molecular characterization ofresistant and accumulating isolated strains

2 Materials and Methods

21 Total Concentration of Heavy Metals of Mining ResiduesSamples The pH and the electrical conductivity (EC) foreach mining residue sample were measured according toAFNOR X31-103 [23] and [24] respectively The total con-centrations of Zn Pb Cd Cu and Cr were determined forall sample residues [23] Samples were oven-dried at 500∘Cfinely ground (2mm) and then ashed in a crucible 5mLof fluohydric acid (50) was added to 05 g of each sampleand brought to dry Samples were then digested with aquaregia (HCl and HNO

3

successively in a ratio of 3 1) Flaskscontaining samples were heated gently until the sampleswere digested which is indicated by the formation of a clearsolution above the residue This solution was adjusted to10mL with distilled water Digested mining residues sampleswere analyzed for metals concentrations (Pb2+ Cu2+ Zn2+Cr6+ and Cd2+) using an UNICAM 929 model flame atomicabsorption spectrophotometer (FAAS)

22 Isolation of Actinobacteria from Mining Residues Tengrams of the residues sample were suspended in 100mLof sterile distilled water and shaken vigorously for 10minAliquots (01mL) of appropriate dilutions were plated onBennettrsquos agar medium [25] Plates were incubated at 30∘Cfor 15 days The isolation and purification of actinobacteriawere done by repeated streaking and dilution-plate tech-niques using Bennettrsquos agar medium All isolates were thenmaintained in glycerol 20 at minus20∘C

23 Selective Medium for the Metal-Resistant ActinobacteriaIn order to choose the appropriatemedium to select themetalresistant actinobacteria two media were used nutrient agar(type Pronadisa) and modified Duxbury agar (KCl 03 gsdotlminus1CaCl2

0025 gsdotlminus1 MgSO4

sdot7H2

O 02 gsdotlminus1 (NH4

)2

SO4

05 gsdotlminus1 glucose 1 gsdotlminus1 tryptone 1 gsdotlminus1 yeast extract05 gsdotlminus1 agar 15 gsdotlminus1) the pH of the medium was adjustedto 700 [26] Tested metals were lead [Pb(NO

3

)2

] copper[Cu(NO

3

)2

sdotH2

O] cadmium [Cd(NO3

)2

sdot4H2

O] chromium[K2

Cr2

O7

] and zinc [Zn(NO3

)2

sdot6H2

O] Stock solutions ofmetals salts prepared in distilled water were sterilized byfiltration (020 120583m) 005mgsdotmLminus1 of each heavy metal wasused in both media (nutrient agar and Duxbury agar) Plateswere incubated at 30∘C for 7 days Growth of actinobacteria

The Scientific World Journal 3

on culture media containing no metals was used ascontrol

24 Screening ofHeavyMetal Resistant Actinobacteria Heavymetal resistance of all actinobacteria isolated from miningresidues was assessed against five elements (Pb Cu Zn Cdand Cr) in triplicate with different concentrations (005 015025 and 1mgsdotmLminus1) in Duxbury agar Growth percentageswere calculated for each metal after comparison with con-trols

To determine the MIC of each metal actinobacteriaisolates were grown in Duxbury agar with increased concen-trations The MIC was defined as the lowest concentration ofthe metal inhibiting the visible growth of the bacteria

25 Screening of Heavy Metal-Accumulating ActinobacteriaActinobacterial isolates were streaked on Duxbury agarplates containing 005mgsdotmLminus1 of each tested metal Afterincubation plates were exposed to hydrogen sulfide gas ina sealed container (resulting from the reaction of Na

2

S withHCl) Simple and effective formation of dark precipitate wasachieved for several metals by treatment with sulfide Uponformation of the metal sulfide plates were carefully screenedto detect any change in the region surrounding bacteriacolonies [27]

In order to determine metal quantities accumulated byisolates actinobacteria strains were cultured on Bennettliquid mediumwithout heavy metals After incubation (30∘Cfor 7 days) cultures were centrifuged at 12000 rpm for20min the pellets were washed twice with phosphate-buffered saline and were resuspended in sterile solutionscontaining 05mgsdotmLminus1 of tested metal The tubes were thenincubated at room temperature in a roller mixer After 3 hof incubation the cells were harvested by centrifugation at12000 rpm for 20min The amount of residual metal presentin the supernatant was measured by an atomic absorptionspectrophotometer At the same time dry weight of thebacterial pellet was determined Values are expressed asthe averages of three determinations carried out in parallelBioaccumulation of the metal in the biomass was expressedas the amount removed from solution containing the testedmetal based on the following equation [3]

Metal removal =[119862

0

minus 119862

119905

] 119881

119872

(1)

where 1198620

is metal initial concentration in the solution(mgsdotmLminus1)119862

119905

is metal concentration after 3 h in the solution(mgsdotmLminus1) 119881 is total volume of the culture and 119872 is drymass of biomass (g)

26 Morphological and Physiological Characterization ofActinobacteria Isolates The morphological cultural phys-iological and biochemical characteristics of the selectedisolates were evaluated as described by Shirling and Gottlieb[28] Cultural characteristics were observed on glycerol-asparagine agar (ISP5) inorganic salts-starch agar (ISP4)tryptone-yeast extract agar (ISP1) and yeast extract-maltextract agar (ISP2) media at 30∘C for 7ndash21 days Melanin

production was detected by growing the isolates on peptone-yeast extract-iron agar (ISP6) [28] The substrate myceliumcolor was identified in terms of Prauserrsquos guide 7 [29]The assimilation of carbohydrates was studied by using themedium ISP9 containing 13 different carbohydrates at aconcentration of 1 (wv) as sole carbon source

27 Molecular Identification of Actinobacteria Isolates TheDNA of selected actinobacteria was isolated according tothe procedure described by Hopwood et al [30] The16S rDNA was amplified using the PCR method with TaqDNA polymerase and primers 27F (51015840-AGAGTTTGA-TCMTGGCTCAG-31015840) and 1492R (51015840-TACGGYTACCTT-GTTACGACTT-31015840) [31] The conditions for thermal cyclingwere as follows denaturation of the target DNA at 98∘Cfor 3min followed by 30 cycles at 94∘C for 1min primerannealing at 52∘C for 1min and primer extension at 72∘C for5min At the end of the cycling the reactionmixturewas heldat 72∘C for 5min and then cooled to 4∘C The PCR productobtained was sequenced using an automated sequencer andthe same primers as above for sequence determination(Macrogen Inc (Seoul Korea)) The sequence was comparedfor similarity with the reference species of bacteria containedin genomic database banks using the NCBI Blast available athttpwwwncbinlmnihgov A phylogenetic tree based on16S rRNA gene sequence was constructed with the neighbor-joining algorithm in MEGA version 5 [32]

28 Siderophore Assays The universal chemical assay ofSchwyn andNeilands [33] was used for detecting siderophoreproduction by actinobacteria using Chrome Azurol S agarassay (CAS) For this experiment glasswares were firstcleaned by immersion in dichromate-acid solution for 48 hand then rinsed several times with distilled water beforeuse In parallel a minimum medium agar (MM) withoutiron (glucose 10 gsdotlminus1 KNO

3


05 gsdotlminus1 KCl05 gsdotlminus1 K

2

HPO4

05 gsdotlminus1 agar 15 gsdotlminus1) was prepared Purestrains of actinobacteria were grown on MM for 3 days at30∘C and then mycelia plugs (9mm in diameter) were cutplaced on CAS agar plates and incubated for 3 days at 30∘CAs control MMwas amended with 001 gsdotlminus1 of FeSO

4

sdot7H2

OA positive reaction is indicated by a color change of CASreagent from blue to orange leading to a clearly visibleyellow-orange halo around the disk

Hydroxamate-specific assay was used to reveal the pres-ence of hydroxamate siderophores in culture supernatantsSiderophore production was monitored by ferric-catalyzedacid hydrolysis and Csaky assay [34] Strains producingsiderophores were inoculated on liquid minimum mediumMM The cultures were incubated for 2 weeks and thencentrifuged at 4000 rpm for 20min at 4∘C the supernatant(2mL) was put in a test tube This sample was saturated withferric ion and a 02mL aliquot was hydrolyzed with 2mLof 3M sulfuric acid for 4 h in a 120∘C (autoclave) Sodiumacetate (2M 7mL) and sulfanilamide (2mL of a 1 [wv]solution in 30 [vv] acetic acid) were added and the pHwasadjusted to 31 plusmn 01 This sample was oxidized with iodine(2mL of 00325 [wv] I

2

in 005 [wv] KI) for 4min at


Table 1 Mean heavy metal concentrations pH and conductivity in mining residues samples

Mining areasMining residues content

Heavy metals content gsdotKgminus1 pH Conductivity (mSsdotcmminus1)Pb Cu Zn Cd Cr

Kettara 0028 plusmn 0007 2942 plusmn 0134 0685 plusmn 0092 ND 0017 plusmn 0002 220 1028Bir Nhass 1407 plusmn 0341 0095 plusmn 0024 9989 plusmn 1223 0075 plusmn 0011 0031 plusmn 0005 652 221Sidi Bouatman 10189 plusmn 3580 0102 plusmn 0029 18870 plusmn 5733 0044 plusmn 0017 0035 plusmn 0005 742 208Arbar plant 1619 plusmn 0432 1243 plusmn 0079 14434 plusmn 0928 0070 plusmn 0007 0006 plusmn 0001 717 227Tenfit mine 0098 plusmn 0002 0498 plusmn 0068 2093 plusmn 1020 ND 0014 plusmn 0001 752 146lowastND not detected

room temperature (24∘C) Excess iodine was removed by theaddition of sodium arsenite (2mL of a 0075 [wv] solutionin distilled water) N-(1-Naphthyl)ethylenediamine (2mL ofa 005 [wv] solution in distilled water) was added andthe mixture was allowed to stand for 30min to complete thecolor development The solution was diluted to 50mL andthe A543

was measured A blank was prepared by addition ofthe reagents to 02mL of distilled water

The presence of catechol siderophores was also investi-gated in culture supernatants using a modified version ofthe Arnow reaction [35 36] In this assay the test sampleis treated with HCl NaNO

2

and Na2

MoO4

-2H2

O whichresults in a yellow color subsequent addition of NaOHcauses the color to change to red in the presence of catecholcompounds Reagents were prepared and reactions carriedout as in Neilands and Nakamura [36] absorbance valueswere determined using a spectrophotometer at a wavelengthof 515 nm

29 Statistical Analysis All experiments were performed intriplicate To compare differences in metal accumulationamong tested isolates obtained data were analyzed throughmultivariate analysis of variance (ANOVA) and the meanswere compared by Tukey multiple range test All numericdifferences in the data were considered significantly differentat the probability level of 119875 le 005

3 Results

31 Assessment of Present Mining Residues Heavy MetalConcentrations The mean metal concentration (in gsdotKgminus1)in all prospected mining residues and their related statisticalparameters is shown in Table 1 Heavy metal concentrationsdid show variation between sites At Sidi Bouatman sitePb and Zn were the dominant elements with 1019 and1887 gsdotKgminus1 respectively While At Arbar plant the dom-inant element was Zn with 1443 gsdotKgminus1 In Kettara mineCu was found to be the dominant metal with 294 gsdotKgminus1for Bir Nhass Zn with 999 gsdotKgminus1 and at Tenfit mine thehigher concentration of the dominant metal (Zn) reached209 gsdotKgminus1 Mean concentrations of Cr and Cd were signif-icantly lower than the other tested metals in all sites Forthe pH and conductivity values there were no significantvariations between the four sites (Bir Nhass Sidi BouatmanArbar plant and Tenfit mine) while a significant decrease

0102030405060708090

100

Pb Cr Cu Zn Cd

Gro

wth

per

cent

ages

of

actin

obac

teria

l iso

late

s

Growth percentages on Duxbury agarGrowth percentages on nutrient agar

Figure 2 Growth percentages of actinobacterial isolates on bothDuxbury agar and nutrient agar with Pb Cu Zn Cd and Crat the concentration 005mgsdotmLminus1 (the percentage of growth wascalculated compared to the control)

in pH values (22) and a significant increase in conductivity(1028 mSsdotcmminus1) were recorded in Kettara site

32 Isolation of Actinobacteria and Screening of Metal-Resistant Strains A total of 59 actinobacteria strains wereisolated from mining residues located in different miningareas around Marrakech city (BN 30 isolates SB 8 isolatesand GT 21 isolates) No actinobacteria strain was isolatedfrom Kettara and Arbar sites

Bacterial resistance to heavy metals was performed intwo culture media (nutrient agar and modified Duxburyagar) Obtained results shown in Figure 2 indicate differencesin growth of strains in both media amended or not withheavy metals (Cd Cu Pb Zn and Cr) In the presenceof metals growth of tested isolates on Duxbury agar wasvery low in comparison with nutrient agar On Duxburyagar supplemented with metals the growth percentagesreached 100 for Pb and Cr while the growth percentagedid not exceed 28 for Cu and 21 for Zn On nutrientagar these percentages were found to be equal to 100for Pb Cu and Cr and 9661 for Zn From these resultsit seems clear that Duxbury agar did not affect or affectsslightly the biodisponibility of metal in the culture medium


Table 2 Heavy metals tolerance in actinobacteria from mining areas

Mining areas Strains Heavy metals mgsdotmLminus1

Pb Cu Zn Cr Cd

Tenfit mine

GT6 GT15 GT39 025 010 010 015 mdashGT14 GT41 010 010 010 010 mdash

GT1 025 010 mdash 015 mdashGT2 GT38 025 010 mdash 010 mdash

GT44 020 010 mdash 010 mdashGT12 GT13 GT3 010 010 mdash 010 mdash

GT4 GT10 030 mdash mdash 010 mdashGT40 025 mdash mdash 010 mdashGT5 020 mdash mdash 010 mdash

GT7 GT8 GT9 GT11 010 mdash mdash 010 mdash

Sidi BouatmanSB20 SB21 030 010 010 010 mdash

SB16 SB18 SB30 SB31 030 mdash 010 015 mdashSB19 SB22 030 mdash mdash 015 mdash

Bir nhass

BN26 030 010 010 015 mdashBN2 055 mdash mdash 015 mdash

BN5 BN9 BN12 BN23 BN46 BN56 BN69 BN71 BN82 030 mdash mdash 015 mdashBN3 BN4 BN13 BN70 BN72 BN73 030 mdash mdash 010 mdash

BN7 BN17 BN22 BN24 BN25 BN27 BN28 BN29 BN68 025 mdash mdash 010 mdashBN47 BN48 BN57 BN58 020 mdash mdash 010 mdash

(mdash) no tolerant strains

For this reason we have chosen Duxbury medium as asuitable medium to select heavy metals resistant actinobacte-ria

Resistance of 59 actinobacteria isolates to the five heavymetals (Pb CuCd Zn andCr)was run andMICvalueswerelisted in Table 2 According to the obtained data maximalMIC values were 055mgsdotmLminus1 for Pb 015mgsdotmLminus1 for Crand 01mgsdotmLminus1 for Zn and Cu for Cd no strain was foundto be resistant

From data in Table 3 differences in heavy metal toxicitylevels against the tested strains were reported One hundredpercent of the tested strains were capable to grow in thepresence of Pb and Cr at 005mgsdotmLminus1 However in thepresence of Cu and Zn at the same concentration only3008 and 3405 of actinobacteria strains were able to growrespectively For lead the percentage of resistant bacteriadecreased with the increase of its concentration in themedium When concentration of Pb reached 025mgsdotmLminus1the percentage of resistant strains decreased to 5540and the growth was completely stopped at 1mgsdotmLminus1 ForCopper Zinc and Chromium actinobacterial growth wascompletely repressed for concentrations greater or equal to015mgsdotmLminus1

From these results lead may be the most tolerated metalby our selected strains and cadmiumwas the most toxic oneChromium zinc and copper gave intermediate results In thisreport we have also noted that resistance percentages to PbZn and Cr among strains isolated from Sidi Bouatman site(Tables 2 and 3) were higher compared to isolates from othersites We can conclude that the resistance levels depended onthe site of strains isolation

33 Screening of Heavy Metal-Accumulating ActinobacteriaAccording to the finding results 27 of the total isolates werefound to be able to accumulate lead on lead-Duxbury agarWe notice the presence of a clear halo around the colony(Figure 3) which shows the disappearance of metal and thismay be due to its accumulation by the strain But none ofstrains was seen to play some accumulation capacities againstCu Cd Cr or Zn

Accumulated quantity results of lead by actinobacterialtested strains are shown in Figure 4 Among the twenty seventested strains the amount of accumulated lead ranged from1298mgsdotgminus1 (BN7) to 61504mgsdotgminus1 (BN3) According tostatistical analysis there was a significant difference (119875 le005) between the accumulation capacity results and thisdifference may depend on tested strain

34 Taxonomic and Molecular Identification of the Actinobac-terial Isolates Selected isolates (27 strains) were tested fortaxonomical diversity using morphological cultural physi-ological and biochemical criteria as well as other features(Table 4) The aerial and substrate mycelium colors weredetermined on ISP2 ISP4 and ISP5 media Tested strainsshowed different abilities to assimilate 13 carbon sources(Table 4) The analysis of 16S rRNA gene sequence of thesestrains (Table 5 and Figure 6) confirmed this classificationPhylogenetic tree based on 16S rRNA gene clearly demon-strated that themajority of selected isolates belong to Strepto-myces genus and only three isolates (GT6 GT15 and GT39)were found belonging to Amycolatopsis genus

The 16S rRNA sequence was deposited in GenBankunder accession numbers KF479164 KF479165 KF479166


Table 3 Growth percentages of actinobacteria isolated from mining areas

Heavy metal Mining areas Number of actinobacteria isolatedConcentration of heavy metals

005 015 025 1mgsdotmLminus1a mgsdotmLminus1a mgsdotmLminus1a mgsdotmLminus1a

Lead

Bir Nhass 30 100 100 5667 mdashSidi Bouatman 8 100 100 100 mdashTenfit mine 21 100 5238 952 mdash

Total 59 100 8413 5540 mdash

Cadmium

Bir Nhass 30 mdash mdash mdash mdashSidi Bouatman 8 mdash mdash mdash mdashTenfit mine 21 mdash mdash mdash mdash

Total 59 mdash mdash mdash mdash

Copper

Bir Nhass 30 333 mdash mdash mdashSidi Bouatman 8 2500 mdash mdash mdashTenfit mine 21 6190 mdash mdash mdash

Total 59 3008 mdash mdash mdash

Zinc



Chromium


Total 59 100 mdash mdash mdashaResults are expressed in percentage of strains grown on culture medium with metal per total tested actinobacteria from each mining residue(mdash) no tolerant strains

Positive lead accumulating actinobacteriapresence of a clear zone indicating an

accumulating activity

Negative lead accumulatingactinobacteria absence of a

clear zone indicating noaccumulating activity

Duxbury agar with lead

Figure 3 Example of positive and negative lead accumulating actinobacteria on Duxbury agar supplemented with Pb

KF479167 KF479168 KF479169 KF479170 KF479171KF479172 KF479173 KF479174 KF479175 KF479176KF479177 KF479178 KF479179 KF479180 KF479181KF479182 KF479183 KF479184 KF479185 KF479186KF479187 KF479188 KF479189 and KF479190 respectivelyfor Streptomyces strains BN2 BN3 BN4 BN7 BN9 BN12BN13 BN17 BN22 BN23 BN24 BN25 BN48 BN68 BN69BN71 BN72 BN73 BN82 GT1 GT2 GT6 GT15 GT39SB22 SB30 and SB31

35 Siderophore Analysis For this parameter 27 lead accu-mulating actinobacterial isolates were checked for sidero-phore secretion Strains were cultivated on CAS medium for

7 days and when a red or orange halo appeared aroundcolonies strain was considered as positive for the secretionof siderophore and was able to chelate iron from the culturemedium (Figure 5) The universal siderophore assay waspositive for only 12 of the 27 tested isolates (BN4 BN17 BN24BN68 BN72 BN82 GT1 GT2 GT6 GT15 SB30 and SB31)

All the 12 siderophore positive strains were able toproduce hydroxamate siderophore type and none of themwas able to produce catecholate siderophore

4 Discussion

The aim of this study was to select actinobacteria strains fromheavy metal contaminated sources of Moroccan abandoned


Table4Biochemicalandmorph

ologicalcharacteris

ticso

f27metalaccumulatingiso

lates

Characteris

tics

Strains

BN3

BN4

BN24

BN68

BN72

BN13

BN17

BN22

BN25

BN48

BN7

BN73

GT1

GT2

SB30

GT15

GT6

GT3

9BN

82BN

2BN

9BN

12BN

69BN

71SB

22BN

23SB

31ISP2

++++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

ISP4

+++

+++

++++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

++++

+++

+++

+++

+++

+ISP5

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

Aria

lspo

remass

WG

Ow

Ow

GW

GG

GG

GV

PiW

CW

WW

WW

WW

WW

WW

CColon

yreverse

BT

YB

BB

TGr

TB

BP

BB

LbB

LbLb

BB

TT

TB

TB

LbSolublep

igment

minusminusminusminusminusminusminusminusminusminusminusminusminusminusminusminusminusminusminusminusminusminusminusminusminusminusminus

Melanin

ontyrosin

eagarminusminusminusminusminusminusminusminusminusminusminusminusminusminusminusminusminusminusminusminusminusminusminusminusminusminusminus

Csource

utilizatio

nSucrose

minusminusminusminusplusmnminusminus

+minus

+++minus

++

+minus

++++

+++

+minusminus

+minus

+++

+++minusminusminus

Fructose

minusminusminusminus

+++

++++minusminus

++++

+++

+++

+++

+++

+++

+++

+++minusminusminusplusmnminus

+++minusminus

Glucose

++

++

++

++

++

++

++

++

++

++

++

++

++

+++

Arabino

seminusminusminusminusminusminusminus

+++minus

++minus

+++

+++minus

+++

+++

+++minusminusminus

++

+minusminus

+Maltose

minusminusminusminusminus

+++

+++

+minus

+++

+++

++++

+++

+minus

+++

+++

+++minusminusminusminusminus

+++minusminus

Manno

seminusminus

++minus

+minusminus

+minusminusminus

+++

++++

++minus

+++

++++

+minusminusplusmn

+++

+minusminusminus

Mannitol

minusminus

++minus

+++

++++

+minus

+++minus

++++

+++

++

++++

+++

+minusminusminusplusmn

+++

+++minusminusminus

Galactose

plusmnplusmnplusmnminusminus

+++

+plusmnminus

+++minus

+++

++++

+++

+++

+++

+minusminusminusplusmnminus

+++minusminus

+Inosito

lminusplusmn

++minusminusminus

+++minus

++++

++++

+++

++

+++

+++

+++minusminusminus

+++

+minusminusminus

+Rh

amno

seminusminusminusminusminusminusminusminusminusminusminusminusminusminusminus

+++

++minusminusminusminus

+minusminusminus

+Sorbito

lminusminusminusminusminus

+minusminusminusminusminus

+++

+minus

+++

+++

+++minusminus

++minus

+plusmnminusminusminus

Lactose

minusminusminusminusminusminus

+minusminus

++

+++

+++

+++

++++

++minusminusminus

++++

+++

+++

++minusminus

Dextrin

minusminusminusminusminusminusminusminusminusminusminusminus

+++

+++minus

++

+minusminusminusminusminusminusminusminus

++ldquo+rdquoT

estedpo

sitiveutilizedas

substrateldquominus

rdquotestednegativ

eno

tutilized

assubstrateldquoplusmn

rdquolow

grow

th

Bbrow

nC

colorle

ssG

grayGrgreen

OwoffwhiteLblight

brow

nP

purpleP

ipink

TtanV

violetWw

hiteY

yello

w


Table 5 Comparison of percent similarities between our 16S rRNA gene sequence and sequences present in the genomic database banksusing NCBI BLAST

Strains Percentage of sequenceidentities () Actinomycetes strains Accession

numberStreptomyces sp BN2Streptomyces sp BN9Streptomyces sp BN12Streptomyces sp BN23Streptomyces sp BN71Streptomyces sp SB22

99Streptomyces sp strain NT307(K3)

Streptomyces albus subsp albus (NRRL B-2365)Streptomyces flocculus (NBRC 13041)

AJ0020831DQ0266691NR 0411001

Streptomyces sp BN7 99 Streptomyces sp HB096Streptomyces diastaticus subsp diastaticus (NBRC 3714)

GU2134921NR0412091

Streptomyces sp BN13Streptomyces sp BN17 99 Streptomyces gougerotii (NBRC 13043)

Streptomyces sp HB096NR1126101GU2134921

Streptomyces sp BN22Streptomyces sp BN25Streptomyces sp BN48

100 Streptomyces sp HB096Streptomyces rutgersensis (NBRC 12819)

GU2134921NR0410771


100Streptomyces sp (VTT E-062974)Streptomyces fungicidicus (YH04)

Streptomyces sp L116

EU4305461AY6361551EU4105091

Streptomyces sp BN4 99 Streptomyces sp (VTT E-062974)Streptomyces sp L116

EU4305461EU4105091

Streptomyces sp BN69 99 Streptomyces albus subsp albus (NRRL B-2365)Streptomyces gibsonii (NBRC 15415)

DQ0266691NR0411801

Streptomyces sp BN72 99 Streptomyces sampsonii strain S151AStreptomyces sp VTT E-062974

HQ4399051EU4305461

Streptomyces sp BN73 100 Streptomyces rochei strain A-1Streptomyces sp (NEAU-L11)

GQ3920581JF5025721

Streptomyces sp BN82 99 Actinobacterium BH0951Streptomyces aurantiogriseus strain NRRL B-5416

GU2657201AY999773

Streptomyces sp GT1Streptomyces sp GT2 99

Streptomyces sp CS38Streptomyces kanamyceticus (NRRL B-2535)

Streptomyces aureus (NBRC 100912)Streptomyces kanamyceticus (NBRC 13414)

EF4942321NR0438221NR1126081NR1123971

Amycolatopsis sp GT6Amycolatopsis sp GT15Amycolatopsis sp GT39

99

Amycolatopsis sp SA08Amycolatopsis decaplanina (DSM 44594)Amycolatopsis japonica (MG 417-CF 17)

Amycolatopsis keratiniphila subsp nogabecina (DSM 44586)Amycolatopsis orientalis (IMSNU 20057)

GU2946851NR0255621NR0255611NR0255621NR0420401

Streptomyces sp SB30Streptomyces sp SB31 99

Streptomyces pluricolorescens (NAPOLSKAYA1 isolate 2)Streptomyces parvus (13647J)

Streptomyces praecox (CGMCC 41782 clone 2)

FR8376311EU7411401JQ9244031

a

b bbc bc

bc bc bc bc bc bcbc bc bc bcdbcd

bcdbcde

cdef

defef ef ef f f f f

0100200300400500600700800

BN3

BN24

BN4

BN82

BN25

BN13

BN69

GT1

5BN

48SB

31BN

17BN

73G

T39

GT6

GT1

BN71

BN23

BN68

SB30

BN12

BN9

GT2

BN2

BN72

SB22

BN22

BN7

Actinobacterial isolates

(mg

of le

admiddotgminus1of

bio

mas

s)

Figure 4 Quantities of removed lead by the tested actinobacteriastrains

minesHeavymetal resistance and accumulation capacities ofselected strains were performed in order to investigate theirapplicability as a bioremediators for polluted areas

Physicochemical analysis of mining residue samplesrevealed the presence of high concentrations of heavy metalsin all studied sites especially for Pb Cu and Zn In gen-eral these results were in accordance with previous studiescarried out in abandoned mines in Morocco [21] and inothers countries such as Sweden Canada and Spain [37ndash40]Excepting Kettara residues samples all samples presented aneutral to slightly alkaline pH explained by the presence ofhigh concentration of carbonates Furthermore the reasonbehind acidic pH inKettaramine is related to the oxidation ofsulfuric acid which in the absence of sufficient neutralizing


Streptomyces sp BN7Streptomyces rutgersensis (NBRC 12819)Streptomyces sp BN22

Streptomyces sp BN17

Streptomyces gougerotii (NBRC 13043)Streptomyces sp BN13

Streptomyces diastaticus subsp diastaticus (NBRC 3714)Streptomyces sp (HB096)Streptomyces sp BN25


Streptomyces sp BN3Streptomyces sp (VTT E-062974)Streptomyces fungicidicus (YH04)Streptomyces sp BN4Streptomyces sp BN24



Streptomyces sampsonii (S151A)Streptomyces rochei (A-1)Streptomyces sp (NEAU-L11)Streptomyces sp BN73


Actinobacterium (BH0951)Streptomyces aurantiogriseus (NRRL B-5416)

Streptomyces sp (CS38)Streptomyces sp GT2Streptomyces sp GT1

Streptomyces aureus (NBRC 100912)Streptomyces kanamyceticus (NRRL B-2535)Streptomyces kanamyceticus (NBRC 13414)

Streptomyces sp SB30Streptomyces sp SB31Streptomyces parvus (13647J)Streptomyces praecox (CGMCC 41782 clone 2)Streptomyces pluricolorescens (NAPOLSKAYA1 isolate 2)Streptomyces badius (CB00830)

Streptomyces albus subsp albus (NRRL B-2365)Streptomyces gibsonii (NBRC 15415)Streptomyces flocculus (NBRC 13041)Streptomyces sp (NT307(K3))Streptomyces sp BN2

Streptomyces sp BN9Streptomyces sp BN12




Streptomyces sp SB22Amycolatopsis orientalis (IMSNU 20057)Amycolatopsis keratiniphila subsp nogabecina (DSM 44586)Amycolatopsis decaplanina (DSM 44594)Amycolatopsis sp (SA08)Amycolatopsis japonica (MG 417-CF 17)


001

Figure 5 Evolutionary relationships of taxa the evolutionary history was inferred using the neighbor-joining method [71] The optimaltree with the sum of branch length = 021501137 is shown The tree is drawn to scale with branch lengths in the same units as those ofthe evolutionary distances used to infer the phylogenetic tree The evolutionary distances were computed using the maximum compositelikelihood method [72] and are in the units of the number of base substitutions per site The analysis involved 55 nucleotide sequencesCodon positions included were 1st + 2nd + 3rd + noncoding All positions containing gaps and missing data were eliminated There were atotal of 1392 positions in the final dataset Evolutionary analyses were conducted in MEGA5 [32] The scale bar represents 001 substitutionsper nucleotide position

components brought down the pH [41ndash43]These proprietiesmay affect directly living bacteriarsquos diversity and ecophysiol-ogy in the prospected sites In the present study the acidicpH may explain why we did not isolate any actinobacteriastrain from the Kettara site Moreover our results showeda negative correlation between actinobacterial diversity andheavy metal pollution levels Indeed we have isolated more

actinobacterial strains in BN (30 isolates) andGT (21 isolates)than in SB site (08 isolates) Several authors pointed out thatheavy metal pollution of aquatic and soil ecosystems inducea decrease in the microbial diversity [5 9] Haferburg andKothe [4] reported that there was a clear mutual influence inmining areas not only are microbes in soil affected by theirenvironment directly and indirectly but also they control


T Control

Positivesiderophoreproducing

strain

Negative siderophoreproducing

strain

Figure 6 Screening of siderophore producing actinobacteria usingchrome azurol S agar (positive strain are yellow-orange negativestrain are blue)

particular soil parameters These authors suggested also thatgrowth and metabolism can lead to changes in pH redoxpotential and ionic strength of the soil

From the prospected mining sites we have isolated atotal of 59 actinobacteria strains All isolates were screenedfor their resistance to Pb Zn Cd Cu and Cr In order toassess the applicability and the suitability of culture mediumin screening heavy metal resistant bacteria we have used twoculture media (rich medium nutrient agar and minimummedium Duxbury agar)

Obtained data revealed that all isolates streaked on nutri-ent agar with metals did not show any significant differencein growth comparing to the control one In contrast asignificant difference in growth was observed for strainstested on Duxbury agar with metals in comparison withthe control We suggest that this is not only due to themetal kind but also due to the culture medium composi-tion Indeed the medium composition may modulate themetal availability and consequently its toxicity Nutrient agarcontaining organic compounds can chelate metals and makeit not available for the tested microorganisms [16 44] Incontrast when isolates were streaked on Duxbury agar withmetals only some of them showed resistance ability So thelatter medium may be considered as a more suitable supportfor such studies Our findings agree with other reports inwhich frequently minimal medium has been used Indeedcomplex media are inadequate due to the high concentrationof organic components that absorb metallic ions Moreoversome heavy metals have high affinity to bind organic matterimplying a decrease of their bioavailability [16 44]The sameobservations were reported by Majzlik et al [45] Schmidtet al [46] reported that a Streptomyces strain grew verydifferently depending on growth media (minimal mediumand TSB complex medium) amended with nickel

Various methods of actinobacteria classification areused elsewhere [47] In several works the combination of

Table 6 Heavymetal concentrations (mgsdotmLminus1) available inminingresidues [21]

Goundafa Sidi Bouatman Bir NhassZn 3600 8406 7671Pb 1280 1922 0065Cu 0022 0023 0110Cd 0043 0174 0054

morphological and chemical properties with 16S rRNAsequencing has been used to differentiate between actino-bacteria genera [48] The 16S rRNA gene sequencing of ourselected isolates demonstrated that 889 of these organismsare phylogenetically related to members of the Streptomycesgenus and 111 are closely related to members of theAmycolatopsis genusAlthough the neighbor-joiningmethodshowed that some isolates belong to the same speciesthey have different physiological and biochemical featuresFurther analyses (as DNA-DNA hybridization) are requiredto clarify the systematic belonging of some of our selectedstrains

It is generally assumed that environmental heavy metalpollution leads to the establishment of a tolerant or resistantmicrobial population [49] In the present study some of thetested strains could resist relatively high concentrations oflead (up to 055mgsdotmLminus1 for Streptomyces sp BN2) and hex-avalent chromium (up to 010mgsdotmLminus1) Polti et al [16] havepreviously isolated nine chromium resistant actinobacteriastrains from contaminated sites in Tucuman (Argentina)which were identified as members of the genus StreptomycesResistance of actinobacteria to Pb was previously reported bySanjenbam et al [50] who have described a maximum toler-ance concentration of Streptomyces that reaches 18mgsdotmLminus1

Our isolates could also resist Cu and Zn (010mgsdotmLminus1)and corroborate those of Albarracın et al [51] who showedthat Amycolatopsis tucumanensis strains isolated fromcopper-polluted sediments were resistant to concentrationsup to 008mgsdotmLminus1 of CuSO

4

In the other hand none ofour isolates were able to grow in the presence of CadmiumA similar observations were recorded for Streptomycescoelicolor A3(2) isolated from an abandoned uraniummining area [46] Amycolatopsis sp GT6 Amycolatopsis spGT15 and Amycolatopsis sp GT39 can tolerate very highconcentrations of the four metals tested (Pb 025 Cu 010Cr 015 and Zn 010mgsdotmLminus1) From obtained resultsit appeared that actinobacterial MICs values which aredetermined with traditional media did not agree with metalconcentrations detected in sites Selected strains are unableto resist to heavy metalrsquos concentrations equal to thoserecorded in mining residue samples This may be explainedby the presence of nonavailable forms of metals in sites withhigh percentages In a previous study on the same sites (SidiBouatman Bir Nhass and Goundafa) heavy metals (PbCu Cd and Zn) are generally unavailable (Table 6) [21]Moreover some interactions can alter chemical speciationand the bioavailability of these micropollutants In the sameway some authors have pointed out that the soluble and


exchangeable fractions of heavy metals largely determinethe availability and mobility of heavy metals [52 53]Furthermore pH is one of the most decisive factors who acton the solubility of heavy metals In addition the presenceof limestone in Goundafa and Sidi Bouatman residues mayexplain the low availability of their toxic form of heavymetals According to these observations we can concludethat our selected isolates had abilities to tolerate relativehigh metal concentrations than what is available in samplingsites This may also explain why all isolates were sensitive tocadmium

Metal accumulation or biotransformation is alternativemechanisms for metal detoxification used by bacteria [54]Chemical precipitation taste of heavy metals using hydrogensulfide technic (H

2

S) revealed that only 27 isolates have astrong ability to accumulate Pb the accumulated concen-trations recorded high levels (up to 600mg of Pb per g ofbiomass for Streptomyces sp BN3) As it had been alreadypointed out by Hernandez et al [27] the technic used todetect heavy metal accumulator actinobacterial strains didgive satisfactory results Obtained results show that selectedisolates are able to accumulate only lead metal SimilarlyRho and Kim [55] reported the capacity of Streptomycesviridochromogenes to accumulate lead up to 164 120583gsdotmgminus1Significantly lead adsorption was much higher for variousfungi including Mucor rouxii (769mgsdotgminus1) [56] and Pae-cilomyces marquandii (324mgsdotgminus1) [57] Streptomyces albuswas also able to adsorb copper and gold as well as lead inits cell wall [58] In contrast A tucumanensis showed thehighest bioaccumulation value for growing cells (25mgsdotgminus1)among other microorganisms previously proposed for biore-mediation processes [51] S zinciresistens accumulated Zn2+(2681mgsdotgminus1) and Cd2+ (85mgsdotgminus1) mainly on the cell wallfollowed by intracellular accumulation [59] The surfaceenvelopes of bacterial cells can adsorb various heavy metalsby means of ionic bonds to their intrinsic chemical groups[55] Anionic groups such as carboxylate and phosphategroups of peptidoglycan and teichoic acids are considered asthe major metal binding sites [55]

The secondary metabolites produced by actinobacteriamay enable the bacteria to cope with stress factors includingtoxic levels of heavy metals [14] Another study revealsthat a Streptomyces strain producing a siderophore wascapable of sequestering a range of ions including Mn CoCd Ni Al Li Cu Zn and Mg [60] In the present studyamong 27 Pb resistant and accumulating strains only 12 werefound to be able to produce siderophore from hydroxamatefamily These findings suggest that there is no relationshipbetween metal accumulation capacities and production ofsiderophore especially in iron rich environsThe exploitationof these isolates in bioremediation of metal contaminatediron deficient soils may be useful tool In the same way someauthors suggested application of these kinds of bacteria as aninterface with plants able to accumulate metals [61 62]

In the other hand we have recorded that Streptomyces spBN2 strain shows a high resistance level to Pb (055mgsdotmLminus1)and a low accumulation capacity of this metal (2637mgsdotgminus1)Streptomyces sp BN3 strain which is twofold less resistant to

Pb than Streptomyces sp BN2 strain was able to accumulatemore than 26 times the quantity of Pb than Streptomycessp BN2 Streptomyces sp BN48 strain showed a low resis-tance level (020mgsdotmLminus1) and presented an intermediateaccumulation capacity (28274mgsdotgminus1) in comparison withStreptomyces sp BN3 and Streptomyces sp BN2 Conse-quently it can be clearly noticed that there was no correlationbetween resistance level and accumulation capacity for testedstrains

In fact pollution levels may influence the genetic make-up of residentmicroorganismsMembers of Streptomyces andAmycolatopsis genera can survive in polluted environmentswith heavy metals as mentioned by Polti et al [16] andAlbarracın et al [17] Alvarez et al [63] reported that the pres-ence of heavy metal resistant strains in different Streptomycesclades may have two different explanations (i) the resistancewas already present in the most recent common ancestor(MRCA) and was then inherited by the different lineages (ii)the different lineages inherited from the MRCA the capacityto develop newmechanisms ormodify existing ones in orderto generate resistance to heavy metals Schutze and Kothe[64] indicated that several morphological physiological andreproductive characteristics of Streptomyces (the filamentousgrowth the formation of hyphae and the production ofspores) would allow its species to occupy extreme environ-ments Since these properties are shared by all species of thegenus this can be interpreted as evidence that supports theinheritance of resistance to heavy metals from the MRCAand this is in agreement with the work done by Zhou et al[65]

Many processes could be used in the bioremediationof contaminated wastewaters and soils Microorganisms asactinobacteria might be a good tool which can be used in abioreactor configuration to treat metal contaminated wastesprior to discharge into the environment Recently reportspointed out that such microorganisms could be used inlarge-scale biofilm bioreactors for the treatment of metal-laden wastewater [66ndash70] In the present study living cellsof actinobacteria gave satisfactorymetal accumulating resultsespecially for leadMore studies should be designed atmolec-ular levels to evaluate and to identify mechanisms involved inthe association between metal resistances and accumulationin order to upgrade the bioremediation potential of thesesstrains These studies seem to be highly promising in termsof the creation of fields that encourage the development ofbioremediation processes using native microorganisms

5 Conclusion

Mining sites are considered suitable for the isolation of heavymetal resistant microorganisms and useful sources of poten-tial bioremediators Many abandoned mining sites are highlycontaminated with metallic elements It is very interesting torestore them especially by combining several environmentfriendly remediation techniques and by improving theirquality In our opinion some of our selected actinobac-teria can play very significant roles in the remediation ofcontaminated sites However further studies are requiredto enhance bioremediation potentialities of our strains to


perform biological process and to optimize their role inremoving heavy metals from contaminated environments

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

References

[1] E Valdman L Erijman F L P Pessoa and S G F LeiteldquoContinuous biosorption of Cu and Zn by immobilized wastebiomass Sargassum sprdquo Process Biochemistry vol 36 no 8-9 pp869ndash873 2001

[2] I H Ceribasi and U Yetis ldquoBiosorption of Ni(ii) and Pb(ii)by Phanerochaete chrysosporium from a binary metal systemmdashkineticsrdquoWater SA vol 27 no 1 pp 15ndash20 2001

[3] S Zafar F Aqil and I Ahmad ldquoMetal tolerance and biosorptionpotential of filamentous fungi isolated from metal contami-nated agricultural soilrdquo Bioresource Technology vol 98 no 13pp 2557ndash2561 2007

[4] G Haferburg and E Kothe ldquoMicrobes and metals interactionsin the environmentrdquo Journal of Basic Microbiology vol 47 no6 pp 453ndash467 2007

[5] L Ezzouhri E Castro M Moya F Espinola and K LairinildquoHeavy metal tolerance of filamentous fungi isolated from pol-luted sites in Tangier MoroccordquoAfrican Journal of MicrobiologyResearch vol 3 no 2 pp 35ndash48 2009

[6] M R Bruins S Kapil and F W Oehme ldquoMicrobial resistancetometals in the environmentrdquo Ecotoxicology and EnvironmentalSafety vol 45 no 3 pp 198ndash207 2000

[7] J Selvin S Shanmugha Priya G Seghal Kiran T Thangaveluand N Sapna Bai ldquoSponge-associated marine bacteria as indi-cators of heavy metal pollutionrdquo Microbiological Research vol164 no 3 pp 352ndash363 2009

[8] S Zapotoczny A Jurkiewicz G Tylko T Anielska and KTurnau ldquoAccumulation of copper by Acremonium pinkerto-niae a fungus isolated from industrial wastesrdquo MicrobiologicalResearch vol 162 no 3 pp 219ndash228 2007

[9] V N Kavamura and E Esposito ldquoBiotechnological strategiesapplied to the decontamination of soils polluted with heavymetalsrdquo Biotechnology Advances vol 28 no 1 pp 61ndash69 2010

[10] J Solecka J Zajko M Postek and A Rajnisz ldquoBiologicallyactive secondary metabolites from Actinomycetesrdquo CentralEuropean Journal of Biology vol 7 no 3 pp 373ndash390 2012

[11] M S Louis J M Benito V H Albarracın T Lebeau MJ Amoroso and C M Abate ldquoHeavy-metal resistant actino-mycetesrdquo inHeavy-Metal Resistant Actinomycetes E LichtfouseJ Schwarzbauer and D Robert Eds pp 757ndash767 SpringerBerlin Germany 2005

[12] J Berdy ldquoBioactive microbial metabolitesrdquo The Journal ofAntibiotics vol 58 no 1 pp 1ndash26 2005

[13] A Schmidt GHaferburgM Sineriz DMertenG Buchel andE Kothe ldquoHeavy metal resistance mechanisms in actinobacte-ria for survival in AMD contaminated soilsrdquoChemie der ErdemdashGeochemistry vol 65 Supplement 1 pp 131ndash144 2005

[14] N-W So J-Y Rho S-Y Lee I C Hancock and J-H KimldquoA lead-absorbing protein with superoxide dismutase activityfrom Streptomyces subrutilusrdquo FEMS Microbiology Letters vol194 no 1 pp 93ndash98 2001

[15] V L Colin L B Villegas and C M Abate ldquoIndigenousmicroorganisms as potential bioremediators for environmentscontaminated with heavy metalsrdquo International Biodeteriora-tion amp Biodegradation vol 69 pp 28ndash37 2012

[16] M A Polti M J Amoroso and C M Abate ldquoChromium(VI)resistance and removal by actinomycete strains isolated fromsedimentsrdquo Chemosphere vol 67 no 4 pp 660ndash667 2007

[17] V H Albarracın P Alonso-Vega M E Trujillo M J Amorosoand C M Abate ldquoAmycolatopsis tucumanensis sp nov acopper-resistant actinobacterium isolated from polluted sed-imentsrdquo International Journal of Systematic and EvolutionaryMicrobiology vol 60 part 2 pp 397ndash401 2010

[18] V H Albarracın B Winik E Kothe M J Amoroso and CM Abate ldquoCopper bioaccumulation by the actinobacteriumAmycolatopsis sp AB0rdquo Journal of Basic Microbiology vol 48no 5 pp 323ndash330 2008

[19] J S D Costa V H Albarracın and C M Abate ldquoResponses ofenvironmental Amycolatopsis strains to copper stressrdquo Ecotoxi-cology and Environmental Safety vol 74 no 7 pp 2020ndash20282011

[20] M S Fuentes C S Benimeli S A Cuozzo and M J AmorosoldquoIsolation of pesticide-degrading actinomycetes from a con-taminated site bacterial growth removal and dechlorination oforganochlorine pesticidesrdquo International Biodeterioration andBiodegradation vol 64 no 6 pp 434ndash441 2010

[21] A EL Gharmali Impact des residus miniers et des eaux resid-uaires sur la contamination metallique des ecosystemes aqua-tiques et terrestres de la region de Marrakech Maroc [PhDthesis] University Cadi Ayyad Marrakech Morocco 2005

[22] M Hakkou M Petrissans I El Bakali P Gerardin and AZoulalian ldquoWettability changes and mass loss during heattreatment ofwoodrdquoHolzforschung vol 59 no 1 pp 35ndash37 2005

[23] AFNOR Quality of Soil Soils Sediments Mise en SolutionTotale par Attaque Acide AFNOR Paris France 1996

[24] G AubertMethods of Soil Analysis GRDP Marseille France1978

[25] K L Jones ldquoFresh isolates of actinomycetes in which thepresence of sporogenousrdquo Journal of Bacteriology vol 57 no 2pp 141ndash145 1949

[26] T Duxbury ldquoToxicity of heavy metals to soil bacteriardquo FEMSMicrobiology Letters vol 11 no 2-3 pp 217ndash220 1981

[27] A Hernandez R P Mellado and J L Martınez ldquoMetalaccumulation and vanadium-induced multidrug resistance byenvironmental isolates of Escherichia hermannii and Enterobac-ter cloacaerdquo Applied and Environmental Microbiology vol 64no 11 pp 4317ndash4320 1998

[28] E B Shirling and D Gottlieb ldquoMethods for characterizationof streptomyces speciesrdquo International Journal of SystematicBacteriology vol 16 no 3 pp 313ndash340 1966

[29] H Prauser ldquoAptness and application of colour codes for exactdescription of colours of Streptomycetesrdquo Zeitschrift fur Allge-meine Mikrobiologie vol 4 no 1 pp 95ndash98 1964

[30] D A Hopwood M J Bibb K F Chater et al Genetic Manip-ulation of Streptomyces A Laboratory Manual The John InnesInstitute Norwich UK 1985

[31] D J Lane ldquo16S23S rRNA sequencingrdquo in Nucleic Acid Tech-niques in Bacterial Systematics E Stackebrandt andMGoodfel-low Eds pp 115ndash175 John Wiley amp Sons New York NY USA1991

[32] K Tamura D Peterson N Peterson G Stecher M Nei andS Kumar ldquoMEGA5 molecular evolutionary genetics analysis


using maximum likelihood evolutionary distance and max-imum parsimony methodsrdquo Molecular Biology and Evolutionvol 28 no 10 pp 2731ndash2739 2011

[33] B Schwyn and J B Neilands ldquoUniversal chemical assay forthe detection and determination of siderophoresrdquo AnalyticalBiochemistry vol 160 no 1 pp 47ndash56 1987

[34] T Z Csaky ldquoOn the estimation of bound hydroxylamine inbiological materialsrdquo Acta Chemica Scandinavica vol 2 pp450ndash454 1948

[35] L E Arnow ldquoColorimetric determination of the components of3 4-dihydroxyphenylalaninemdashtyrosine mixturesrdquo The Journalof Biological Chemistry vol 118 pp 531ndash537 1937

[36] J B Neilands and K Nakamura ldquoDetection determinationisolation characterization and regulation of microbial ironchelatesrdquo in CRC Handbook of Microbial Iron Chelates GWinkelmann Ed pp 1ndash14 CRC Press Boca Raton Fla USA1991

[37] H Holmstrom J Ljungberg and B Ohlander ldquoThe characterof the suspended and dissolved phases in the water cover of theflooded mine tailings at Stekenjokk Northern Swedenrdquo Scienceof the Total Environment vol 247 no 1 pp 15ndash31 2000

[38] H Holmstrom and B Ohlander ldquoLayers rich in Fe- and Mn-oxyhydroxides formed at the tailings-pond water interface apossible trap for trace metals in flooded mine tailingsrdquo Journalof Geochemical Exploration vol 74 no 1ndash3 pp 189ndash203 2001

[39] J Ljungberg and B Ohlander ldquoThe geochemical dynamics ofoxidising mine tailings at Laver Northern Swedenrdquo Journal ofGeochemical Exploration vol 74 no 1ndash3 pp 57ndash72 2001

[40] B Vigneault P G C Campbell A Tessier and R de VitreldquoGeochemical changes in sulfidic mine tailings stored under ashallow water coverrdquo Water Research vol 35 no 4 pp 1066ndash1076 2001

[41] S R Jennings D J Dollhopf and W P Inskeep ldquoAcid produc-tion from sulfide minerals using hydrogen peroxide weather-ingrdquo Applied Geochemistry vol 15 no 2 pp 235ndash243 2000

[42] A Khalil L Hanich R Hakkou and M Lepage ldquoGIS-basedenvironmental database for assessing the mine pollution acase study of an abandoned mine site in Moroccordquo Journal ofGeochemical Exploration vol 144 part C pp 468ndash477 2014

[43] M Lghoul A Maqsoud R Hakkou and A Kchikach ldquoHydro-geochemical behavior around the abandoned Kettara mine siteMoroccordquo Journal of Geochemical Exploration vol 144 part Cpp 456ndash467 2013

[44] R S Laxman and S More ldquoReduction of hexavalent chromiumby Streptomyces griseusrdquoMinerals Engineering vol 15 no 11 pp831ndash837 2002

[45] P Majzlik A Strasky V Adam et al ldquoInfluence of zinc(II) andcopper(II) ions on Streptomyces bacteria revealed by electro-chemistryrdquo International Journal of Electrochemical Science vol6 no 6 pp 2171ndash2191 2011

[46] A Schmidt G Haferburg U Lischke et al ldquoHeavy metalresistance to the extreme streptomyces strains from a formeruraniummining areardquo Chemie der ErdemdashGeochemistry vol 69supplement 2 pp 35ndash44 2009

[47] J G Holt N R Krieg P H A Sneath J T Staley and ST Williams Bergeyrsquos Manual of Determinative BacteriologyWilliams ampWilliams Baltimore Md USA 9th edition 1994

[48] E Stackebrandt F A Rainey and N LWard-Rainey ldquoProposalfor a new hierarchic classification system Actinobacteria classisnovrdquo International Journal of Systematic Bacteriology vol 47 no2 pp 479ndash491 1997

[49] AAleem J Isar andAMalik ldquoImpact of long-term applicationof industrial wastewater on the emergence of resistance traitsin Azotobacter chroococcum isolated from rhizospheric soilrdquoBioresource Technology vol 86 no 1 pp 7ndash13 2003

[50] P Sanjenbam K Saurav and K Kannabiran ldquoBiosorptionof mercury and lead by aqueous Streptomyces VITSVK9 spisolated from marine sediments from the bay of Bengal IndiardquoFrontiers of Chemical Science and Engineering vol 6 no 2 pp198ndash202 2012

[51] V H Albarracın M J Amoroso and C M Abate ldquoIsola-tion and characterization of indigenous copper-resistant acti-nomycete strainsrdquo Chemie der ErdemdashGeochemistry vol 65Supplement 1 pp 145ndash156 2005

[52] Y Ge PMurray andWHHendershot ldquoTracemetal speciationand bioavailability in urban soilsrdquo Environmental Pollution vol107 no 1 pp 137ndash144 2000

[53] C E Martınez and H L Motto ldquoSolubility of lead zinc andcopper added to mineral soilsrdquo Environmental Pollution vol107 no 1 pp 153ndash158 2000

[54] G M Gadd ldquoMetals and microorganisms a problem of defini-tionrdquo FEMS Microbiology Letters vol 100 no 1ndash3 pp 197ndash2031992

[55] J Y Rho and J H Kim ldquoHeavy metal biosorption and itssignificance to metal tolerance of streptomycetesrdquo The Journalof Microbiology vol 40 no 1 pp 51ndash54 2002

[56] W Lo H Chua K-H Lam and S-P Bi ldquoA comparativeinvestigation on the biosorption of lead by filamentous fungalbiomassrdquo Chemosphere vol 39 no 15 pp 2723ndash2736 1999

[57] M Słaba and J Długonski ldquoZinc and lead uptake by myceliumand regenerating protoplasts ofVerticilliummarquandiirdquoWorldJournal of Microbiology and Biotechnology vol 20 no 3 pp323ndash328 2004

[58] B Mattuschka G Straube and J T Trevors ldquoSilver copperlead and zinc accumulation byPseudomonas stutzeriAG259 andStreptomyces albus electron microscopy and energy dispersiveX-ray studiesrdquo BioMetals vol 7 no 2 pp 201ndash208 1994

[59] Y Lin X Wang B Wang O Mohamad and G Wei ldquoBioac-cumulation characterization of zinc and cadmium by Strepto-myces zinciresistens a novel actinomyceterdquo Ecotoxicology andEnvironmental Safety vol 77 pp 7ndash17 2012

[60] I Nakouti and G Hobbs ldquoA new approach to studying ionuptake by actinomycetesrdquo Journal of Basic Microbiology vol 53no 11 pp 913ndash916 2013

[61] W Wang Z Qiu H Tan and L Cao ldquoSiderophore productionby actinobacteriardquo BioMetals vol 27 no 4 pp 623ndash631 2014

[62] T Gaonkar and S Bhosle ldquoEffect of metals on a siderophoreproducing bacterial isolate and its implications on microbialassisted bioremediation of metal contaminated soilsrdquo Chemo-sphere vol 93 no 9 pp 1835ndash1843 2013

[63] A Alvarez S A Catalano and M J Amoroso ldquoHeavy metalresistant strains are widespread along Streptomyces phylogenyrdquoMolecular Phylogenetics and Evolution vol 66 no 3 pp 1083ndash1088 2013

[64] E Schutze and E Kothe ldquoBio-geo interactions in metal-contaminated soilsrdquo in Soil Biology E Kothe and A VarmaEds vol 31 pp 163ndash182 Springer Berlin Germany 2012

[65] M Zhou X Jing P Xie et al ldquoSequential deletion of allthe polyketide synthase and nonribosomal peptide synthetasebiosynthetic gene clusters and a 900-kb subtelomeric sequenceof the linear chromosome of Streptomyces coelicolorrdquo FEMSMicrobiology Letters vol 333 no 2 pp 169ndash179 2012


[66] L Diels P H Spaans S van Roy et al ldquoHeavy metals removalby sand filters inoculated with metal sorbing and precipitatingbacteriardquo Hydrometallurgy vol 71 no 1-2 pp 235ndash241 2003

[67] A Ganguli and A K Tripathi ldquoBioremediation of toxic chro-mium from electroplating effluent by chromate-reducing Pseu-domonas aeruginosa A2Chr in two bioreactorsrdquo Applied Micro-biology and Biotechnology vol 58 no 3 pp 416ndash420 2002

[68] A Malik ldquoMetal bioremediation through growing cellsrdquo Envi-ronment International vol 30 no 2 pp 261ndash278 2004

[69] W-L Smith ldquoHexavalent chromium reduction and precipi-tation by sulphate-reducing bacterial biofilmsrdquo EnvironmentalGeochemistry and Health vol 23 no 3 pp 297ndash300 2001

[70] A S Y Ting and C C Choong ldquoBioaccumulation and biosorp-tion efficacy of Trichoderma isolate SP2F1 in removing copper(Cu(II)) from aqueous solutionsrdquoWorld Journal ofMicrobiologyand Biotechnology vol 25 no 8 pp 1431ndash1437 2009

[71] N Saitou and M Nei ldquoThe neighbor-joining method a newmethod for reconstructing phylogenetic treesrdquo Molecular Biol-ogy and Evolution vol 4 no 4 pp 406ndash425 1987

[72] K Tamura M Nei and S Kumar ldquoProspects for inferringvery large phylogenies by using the neighbor-joining methodrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 101 no 30 pp 11030ndash11035 2004

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


Studied mining siteAffluents of the Tensiftriver Road Goundafa

Haut Atlas mountain

Marrakech

Tensift river

KettaraMassif Jbilet Sidi

BouatmanBir Nhass

NN

Marrakech

Rabat

25km

500km

Figure 1 Localisation of prospected abandoned mining areas ofMarrakech Morocco Kettara Sidi bouatman Bir Nhass andGoundafa

reduce or to transform toxic contaminants into their less toxicforms Actinobacteria constitute a morphologically diversegroup of Gram-positive bacteria with high metabolic ver-satility [10] As reported earlier actinomycetes are the goodbioremediation [11ndash16] Their metabolic diversity particulargrowth characteristics mycelial form and relatively rapidability to colonize selective substrates make them well-suitedfor use as agents for bioremediation of inorganic and organiccompounds [16ndash20]

Industrial and mining activities are important for eco-nomic development especially in many developing countriesasMorocco However these activities also represent the mainsources of heavy metal contamination

InMarrakech regionmany exploitationmetalmines haveceased to be functional many years ago Mining residueswere abandoned in open air without any treatment and wereexposed to the atmospheric hazards In fact these releaseheavy metals and other pollutants to the drainage and rep-resent a potential danger of surrounding terrestrial andaquatic ecosystemsMoreover thesemining sites are generallysituated near agricultural zones [21]

To our knowledge little research had been done regardingthe problematic of mining residues in our country despiteawareness of health and environmental risks that they maycause

In the present study we are interested in mining residuescollected from various abandoned mining areas locatedaround Marrakech region West Central Morocco (Figure 1)Kettara mine (KT) is located approximately 30 km North-West of Marrakech and was operated for pyrrhotine extrac-tion until 1981 [22] Sidi Bouatman mine (SB) is locatedapproximately 33 km north of Marrakech and was operatedfor Pb Zn and pyrite extraction until 1980 The old Bir

Nhass zinc mine (BN) is located approximately 30 km northof Marrakech it was closed at the end of the 20th centuryGoundafa mine located approximately at 90 km south ofMarrakech operated for Pb Zn and Cu extraction and in1989 the mine has ceased to be functional In Goundafa areasamples were collected from two sites Arbar plant (GA) andTenfit Mine (GT)

The aim of the present work deals with the evaluation ofthe contamination levels of mining residues the isolation ofheavymetals resistant actinobacteria their potential capacityto accumulate heavy metals (Pb Cu Zn Cd and Cr)and both morphological and molecular characterization ofresistant and accumulating isolated strains

2 Materials and Methods

21 Total Concentration of Heavy Metals of Mining ResiduesSamples The pH and the electrical conductivity (EC) foreach mining residue sample were measured according toAFNOR X31-103 [23] and [24] respectively The total con-centrations of Zn Pb Cd Cu and Cr were determined forall sample residues [23] Samples were oven-dried at 500∘Cfinely ground (2mm) and then ashed in a crucible 5mLof fluohydric acid (50) was added to 05 g of each sampleand brought to dry Samples were then digested with aquaregia (HCl and HNO

3

successively in a ratio of 3 1) Flaskscontaining samples were heated gently until the sampleswere digested which is indicated by the formation of a clearsolution above the residue This solution was adjusted to10mL with distilled water Digested mining residues sampleswere analyzed for metals concentrations (Pb2+ Cu2+ Zn2+Cr6+ and Cd2+) using an UNICAM 929 model flame atomicabsorption spectrophotometer (FAAS)

22 Isolation of Actinobacteria from Mining Residues Tengrams of the residues sample were suspended in 100mLof sterile distilled water and shaken vigorously for 10minAliquots (01mL) of appropriate dilutions were plated onBennettrsquos agar medium [25] Plates were incubated at 30∘Cfor 15 days The isolation and purification of actinobacteriawere done by repeated streaking and dilution-plate tech-niques using Bennettrsquos agar medium All isolates were thenmaintained in glycerol 20 at minus20∘C

23 Selective Medium for the Metal-Resistant ActinobacteriaIn order to choose the appropriatemedium to select themetalresistant actinobacteria two media were used nutrient agar(type Pronadisa) and modified Duxbury agar (KCl 03 gsdotlminus1CaCl2


sdot7H2

O 02 gsdotlminus1 (NH4

)2

SO4

05 gsdotlminus1 glucose 1 gsdotlminus1 tryptone 1 gsdotlminus1 yeast extract05 gsdotlminus1 agar 15 gsdotlminus1) the pH of the medium was adjustedto 700 [26] Tested metals were lead [Pb(NO

3

)2

] copper[Cu(NO

3

)2

sdotH2

O] cadmium [Cd(NO3

)2

sdot4H2

O] chromium[K2

Cr2

O7

] and zinc [Zn(NO3

)2

sdot6H2

O] Stock solutions ofmetals salts prepared in distilled water were sterilized byfiltration (020 120583m) 005mgsdotmLminus1 of each heavy metal wasused in both media (nutrient agar and Duxbury agar) Plateswere incubated at 30∘C for 7 days Growth of actinobacteria






2




0

minus 119862

119905

] 119881

119872

(1)

where 1198620


119905






3



2

HPO4


4

sdot7H2



2










2

and Na2

MoO4

-2H2



3 Results


0102030405060708090

100

Pb Cr Cu Zn Cd

Gro

wth

per

cent

ages

of

actin

obac

teria

l iso

late

s









Pb Cu Zn Cr Cd

Tenfit mine








Bir nhass

















Lead


Total 59 100 8413 5540 mdash

Cadmium



Copper



Zinc



Chromium













4 Discussion




ologicalcharacteris

ticso


lates

Characteris

tics

Strains

BN3

BN4

BN24

BN68

BN72

BN13

BN17

BN22

BN25

BN48

BN7

BN73

GT1

GT2

SB30

GT15

GT6

GT3

9BN

82BN

2BN

9BN

12BN

69BN

71SB

22BN

23SB

31ISP2

++++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

ISP4

+++

+++

++++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

++++

+++

+++

+++

+++

+ISP5

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

Aria

lspo

remass

WG

Ow

Ow

GW

GG

GG

GV

PiW

CW

WW

WW

WW

WW

WW

CColon

yreverse

BT

YB

BB

TGr

TB

BP

BB

LbB

LbLb

BB

TT

TB

TB

LbSolublep

igment


Melanin

ontyrosin


Csource

utilizatio

nSucrose


+minus

+++minus

++

+minus

++++

+++

+minusminus

+minus

+++

+++minusminusminus

Fructose


+++

++++minusminus

++++

+++

+++

+++

+++

+++

+++


+++minusminus

Glucose

++

++

++

++

++

++

++

++

++

++

++

++

++

+++

Arabino


+++minus

++minus

+++

+++minus

+++

+++

+++minusminusminus

++

+minusminus

+Maltose


+++

+++

+minus

+++

+++

++++

+++

+minus

+++

+++


+++minusminus

Manno

seminusminus

++minus

+minusminus

+minusminusminus

+++

++++

++minus

+++

++++

+minusminusplusmn

+++

+minusminusminus

Mannitol

minusminus

++minus

+++

++++

+minus

+++minus

++++

+++

++

++++

+++


+++

+++minusminusminus

Galactose


+++

+plusmnminus

+++minus

+++

++++

+++

+++

+++


+++minusminus

+Inosito

lminusplusmn

++minusminusminus

+++minus

++++

++++

+++

++

+++

+++

+++minusminusminus

+++

+minusminusminus

+Rh

amno


+++


+minusminusminus

+Sorbito



+++

+minus

+++

+++

+++minusminus

++minus


Lactose


+minusminus

++

+++

+++

+++

++++

++minusminusminus

++++

+++

+++

++minusminus

Dextrin


+++

+++minus

++


++ldquo+rdquoT

estedpo

sitiveutilizedas

substrateldquominus

rdquotestednegativ

eno

tutilized


rdquolow

grow

th

Bbrow

nC

colorle

ssG

grayGrgreen

OwoffwhiteLblight

brow

nP

purpleP

ipink

TtanV

violetWw

hiteY

yello

w







AJ0020831DQ0266691NR 0411001


GU2134921NR0412091





GU2134921NR0410771




EU4305461AY6361551EU4105091


EU4305461EU4105091


DQ0266691NR0411801


HQ4399051EU4305461


GQ3920581JF5025721


GU2657201AY999773




EF4942321NR0438221NR1126081NR1123971


99



GU2946851NR0255621NR0255611NR0255621NR0420401




FR8376311EU7411401JQ9244031

a

b bbc bc


bcdbcde

cdef

defef ef ef f f f f

0100200300400500600700800

BN3

BN24

BN4

BN82

BN25

BN13

BN69

GT1

5BN

48SB

31BN

17BN

73G

T39

GT6

GT1

BN71

BN23

BN68

SB30

BN12

BN9

GT2

BN2

BN72

SB22

BN22

BN7


(mg

of le

admiddotgminus1of

bio

mas

s)


























001





T Control


strain


strain











4





2







5 Conclusion






References



















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






2




0

minus 119862

119905

] 119881

119872

(1)

where 1198620


119905






3



2

HPO4


4

sdot7H2



2










2

and Na2

MoO4

-2H2



3 Results


0102030405060708090

100

Pb Cr Cu Zn Cd

Gro

wth

per

cent

ages

of

actin

obac

teria

l iso

late

s









Pb Cu Zn Cr Cd

Tenfit mine








Bir nhass

















Lead


Total 59 100 8413 5540 mdash

Cadmium



Copper



Zinc



Chromium













4 Discussion




ologicalcharacteris

ticso


lates

Characteris

tics

Strains

BN3

BN4

BN24

BN68

BN72

BN13

BN17

BN22

BN25

BN48

BN7

BN73

GT1

GT2

SB30

GT15

GT6

GT3

9BN

82BN

2BN

9BN

12BN

69BN

71SB

22BN

23SB

31ISP2

++++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

ISP4

+++

+++

++++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

++++

+++

+++

+++

+++

+ISP5

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

Aria

lspo

remass

WG

Ow

Ow

GW

GG

GG

GV

PiW

CW

WW

WW

WW

WW

WW

CColon

yreverse

BT

YB

BB

TGr

TB

BP

BB

LbB

LbLb

BB

TT

TB

TB

LbSolublep

igment


Melanin

ontyrosin


Csource

utilizatio

nSucrose


+minus

+++minus

++

+minus

++++

+++

+minusminus

+minus

+++

+++minusminusminus

Fructose


+++

++++minusminus

++++

+++

+++

+++

+++

+++

+++


+++minusminus

Glucose

++

++

++

++

++

++

++

++

++

++

++

++

++

+++

Arabino


+++minus

++minus

+++

+++minus

+++

+++

+++minusminusminus

++

+minusminus

+Maltose


+++

+++

+minus

+++

+++

++++

+++

+minus

+++

+++


+++minusminus

Manno

seminusminus

++minus

+minusminus

+minusminusminus

+++

++++

++minus

+++

++++

+minusminusplusmn

+++

+minusminusminus

Mannitol

minusminus

++minus

+++

++++

+minus

+++minus

++++

+++

++

++++

+++


+++

+++minusminusminus

Galactose


+++

+plusmnminus

+++minus

+++

++++

+++

+++

+++


+++minusminus

+Inosito

lminusplusmn

++minusminusminus

+++minus

++++

++++

+++

++

+++

+++

+++minusminusminus

+++

+minusminusminus

+Rh

amno


+++


+minusminusminus

+Sorbito



+++

+minus

+++

+++

+++minusminus

++minus


Lactose


+minusminus

++

+++

+++

+++

++++

++minusminusminus

++++

+++

+++

++minusminus

Dextrin


+++

+++minus

++


++ldquo+rdquoT

estedpo

sitiveutilizedas

substrateldquominus

rdquotestednegativ

eno

tutilized


rdquolow

grow

th

Bbrow

nC

colorle

ssG

grayGrgreen

OwoffwhiteLblight

brow

nP

purpleP

ipink

TtanV

violetWw

hiteY

yello

w







AJ0020831DQ0266691NR 0411001


GU2134921NR0412091





GU2134921NR0410771




EU4305461AY6361551EU4105091


EU4305461EU4105091


DQ0266691NR0411801


HQ4399051EU4305461


GQ3920581JF5025721


GU2657201AY999773




EF4942321NR0438221NR1126081NR1123971


99



GU2946851NR0255621NR0255611NR0255621NR0420401




FR8376311EU7411401JQ9244031

a

b bbc bc


bcdbcde

cdef

defef ef ef f f f f

0100200300400500600700800

BN3

BN24

BN4

BN82

BN25

BN13

BN69

GT1

5BN

48SB

31BN

17BN

73G

T39

GT6

GT1

BN71

BN23

BN68

SB30

BN12

BN9

GT2

BN2

BN72

SB22

BN22

BN7


(mg

of le

admiddotgminus1of

bio

mas

s)


























001





T Control


strain


strain











4





2







5 Conclusion






References



















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology









2

and Na2

MoO4

-2H2



3 Results


0102030405060708090

100

Pb Cr Cu Zn Cd

Gro

wth

per

cent

ages

of

actin

obac

teria

l iso

late

s









Pb Cu Zn Cr Cd

Tenfit mine








Bir nhass

















Lead


Total 59 100 8413 5540 mdash

Cadmium



Copper



Zinc



Chromium













4 Discussion




ologicalcharacteris

ticso


lates

Characteris

tics

Strains

BN3

BN4

BN24

BN68

BN72

BN13

BN17

BN22

BN25

BN48

BN7

BN73

GT1

GT2

SB30

GT15

GT6

GT3

9BN

82BN

2BN

9BN

12BN

69BN

71SB

22BN

23SB

31ISP2

++++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

ISP4

+++

+++

++++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

++++

+++

+++

+++

+++

+ISP5

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

Aria

lspo

remass

WG

Ow

Ow

GW

GG

GG

GV

PiW

CW

WW

WW

WW

WW

WW

CColon

yreverse

BT

YB

BB

TGr

TB

BP

BB

LbB

LbLb

BB

TT

TB

TB

LbSolublep

igment


Melanin

ontyrosin


Csource

utilizatio

nSucrose


+minus

+++minus

++

+minus

++++

+++

+minusminus

+minus

+++

+++minusminusminus

Fructose


+++

++++minusminus

++++

+++

+++

+++

+++

+++

+++


+++minusminus

Glucose

++

++

++

++

++

++

++

++

++

++

++

++

++

+++

Arabino


+++minus

++minus

+++

+++minus

+++

+++

+++minusminusminus

++

+minusminus

+Maltose


+++

+++

+minus

+++

+++

++++

+++

+minus

+++

+++


+++minusminus

Manno

seminusminus

++minus

+minusminus

+minusminusminus

+++

++++

++minus

+++

++++

+minusminusplusmn

+++

+minusminusminus

Mannitol

minusminus

++minus

+++

++++

+minus

+++minus

++++

+++

++

++++

+++


+++

+++minusminusminus

Galactose


+++

+plusmnminus

+++minus

+++

++++

+++

+++

+++


+++minusminus

+Inosito

lminusplusmn

++minusminusminus

+++minus

++++

++++

+++

++

+++

+++

+++minusminusminus

+++

+minusminusminus

+Rh

amno


+++


+minusminusminus

+Sorbito



+++

+minus

+++

+++

+++minusminus

++minus


Lactose


+minusminus

++

+++

+++

+++

++++

++minusminusminus

++++

+++

+++

++minusminus

Dextrin


+++

+++minus

++


++ldquo+rdquoT

estedpo

sitiveutilizedas

substrateldquominus

rdquotestednegativ

eno

tutilized


rdquolow

grow

th

Bbrow

nC

colorle

ssG

grayGrgreen

OwoffwhiteLblight

brow

nP

purpleP

ipink

TtanV

violetWw

hiteY

yello

w







AJ0020831DQ0266691NR 0411001


GU2134921NR0412091





GU2134921NR0410771




EU4305461AY6361551EU4105091


EU4305461EU4105091


DQ0266691NR0411801


HQ4399051EU4305461


GQ3920581JF5025721


GU2657201AY999773




EF4942321NR0438221NR1126081NR1123971


99



GU2946851NR0255621NR0255611NR0255621NR0420401




FR8376311EU7411401JQ9244031

a

b bbc bc


bcdbcde

cdef

defef ef ef f f f f

0100200300400500600700800

BN3

BN24

BN4

BN82

BN25

BN13

BN69

GT1

5BN

48SB

31BN

17BN

73G

T39

GT6

GT1

BN71

BN23

BN68

SB30

BN12

BN9

GT2

BN2

BN72

SB22

BN22

BN7


(mg

of le

admiddotgminus1of

bio

mas

s)


























001





T Control


strain


strain











4





2







5 Conclusion






References



















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




Pb Cu Zn Cr Cd

Tenfit mine








Bir nhass

















Lead


Total 59 100 8413 5540 mdash

Cadmium



Copper



Zinc



Chromium













4 Discussion




ologicalcharacteris

ticso


lates

Characteris

tics

Strains

BN3

BN4

BN24

BN68

BN72

BN13

BN17

BN22

BN25

BN48

BN7

BN73

GT1

GT2

SB30

GT15

GT6

GT3

9BN

82BN

2BN

9BN

12BN

69BN

71SB

22BN

23SB

31ISP2

++++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

ISP4

+++

+++

++++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

++++

+++

+++

+++

+++

+ISP5

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

Aria

lspo

remass

WG

Ow

Ow

GW

GG

GG

GV

PiW

CW

WW

WW

WW

WW

WW

CColon

yreverse

BT

YB

BB

TGr

TB

BP

BB

LbB

LbLb

BB

TT

TB

TB

LbSolublep

igment


Melanin

ontyrosin


Csource

utilizatio

nSucrose


+minus

+++minus

++

+minus

++++

+++

+minusminus

+minus

+++

+++minusminusminus

Fructose


+++

++++minusminus

++++

+++

+++

+++

+++

+++

+++


+++minusminus

Glucose

++

++

++

++

++

++

++

++

++

++

++

++

++

+++

Arabino


+++minus

++minus

+++

+++minus

+++

+++

+++minusminusminus

++

+minusminus

+Maltose


+++

+++

+minus

+++

+++

++++

+++

+minus

+++

+++


+++minusminus

Manno

seminusminus

++minus

+minusminus

+minusminusminus

+++

++++

++minus

+++

++++

+minusminusplusmn

+++

+minusminusminus

Mannitol

minusminus

++minus

+++

++++

+minus

+++minus

++++

+++

++

++++

+++


+++

+++minusminusminus

Galactose


+++

+plusmnminus

+++minus

+++

++++

+++

+++

+++


+++minusminus

+Inosito

lminusplusmn

++minusminusminus

+++minus

++++

++++

+++

++

+++

+++

+++minusminusminus

+++

+minusminusminus

+Rh

amno


+++


+minusminusminus

+Sorbito



+++

+minus

+++

+++

+++minusminus

++minus


Lactose


+minusminus

++

+++

+++

+++

++++

++minusminusminus

++++

+++

+++

++minusminus

Dextrin


+++

+++minus

++


++ldquo+rdquoT

estedpo

sitiveutilizedas

substrateldquominus

rdquotestednegativ

eno

tutilized


rdquolow

grow

th

Bbrow

nC

colorle

ssG

grayGrgreen

OwoffwhiteLblight

brow

nP

purpleP

ipink

TtanV

violetWw

hiteY

yello

w







AJ0020831DQ0266691NR 0411001


GU2134921NR0412091





GU2134921NR0410771




EU4305461AY6361551EU4105091


EU4305461EU4105091


DQ0266691NR0411801


HQ4399051EU4305461


GQ3920581JF5025721


GU2657201AY999773




EF4942321NR0438221NR1126081NR1123971


99



GU2946851NR0255621NR0255611NR0255621NR0420401




FR8376311EU7411401JQ9244031

a

b bbc bc


bcdbcde

cdef

defef ef ef f f f f

0100200300400500600700800

BN3

BN24

BN4

BN82

BN25

BN13

BN69

GT1

5BN

48SB

31BN

17BN

73G

T39

GT6

GT1

BN71

BN23

BN68

SB30

BN12

BN9

GT2

BN2

BN72

SB22

BN22

BN7


(mg

of le

admiddotgminus1of

bio

mas

s)


























001





T Control


strain


strain











4





2







5 Conclusion






References



















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





Lead


Total 59 100 8413 5540 mdash

Cadmium



Copper



Zinc



Chromium













4 Discussion




ologicalcharacteris

ticso


lates

Characteris

tics

Strains

BN3

BN4

BN24

BN68

BN72

BN13

BN17

BN22

BN25

BN48

BN7

BN73

GT1

GT2

SB30

GT15

GT6

GT3

9BN

82BN

2BN

9BN

12BN

69BN

71SB

22BN

23SB

31ISP2

++++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

ISP4

+++

+++

++++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

++++

+++

+++

+++

+++

+ISP5

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

Aria

lspo

remass

WG

Ow

Ow

GW

GG

GG

GV

PiW

CW

WW

WW

WW

WW

WW

CColon

yreverse

BT

YB

BB

TGr

TB

BP

BB

LbB

LbLb

BB

TT

TB

TB

LbSolublep

igment


Melanin

ontyrosin


Csource

utilizatio

nSucrose


+minus

+++minus

++

+minus

++++

+++

+minusminus

+minus

+++

+++minusminusminus

Fructose


+++

++++minusminus

++++

+++

+++

+++

+++

+++

+++


+++minusminus

Glucose

++

++

++

++

++

++

++

++

++

++

++

++

++

+++

Arabino


+++minus

++minus

+++

+++minus

+++

+++

+++minusminusminus

++

+minusminus

+Maltose


+++

+++

+minus

+++

+++

++++

+++

+minus

+++

+++


+++minusminus

Manno

seminusminus

++minus

+minusminus

+minusminusminus

+++

++++

++minus

+++

++++

+minusminusplusmn

+++

+minusminusminus

Mannitol

minusminus

++minus

+++

++++

+minus

+++minus

++++

+++

++

++++

+++


+++

+++minusminusminus

Galactose


+++

+plusmnminus

+++minus

+++

++++

+++

+++

+++


+++minusminus

+Inosito

lminusplusmn

++minusminusminus

+++minus

++++

++++

+++

++

+++

+++

+++minusminusminus

+++

+minusminusminus

+Rh

amno


+++


+minusminusminus

+Sorbito



+++

+minus

+++

+++

+++minusminus

++minus


Lactose


+minusminus

++

+++

+++

+++

++++

++minusminusminus

++++

+++

+++

++minusminus

Dextrin


+++

+++minus

++


++ldquo+rdquoT

estedpo

sitiveutilizedas

substrateldquominus

rdquotestednegativ

eno

tutilized


rdquolow

grow

th

Bbrow

nC

colorle

ssG

grayGrgreen

OwoffwhiteLblight

brow

nP

purpleP

ipink

TtanV

violetWw

hiteY

yello

w







AJ0020831DQ0266691NR 0411001


GU2134921NR0412091





GU2134921NR0410771




EU4305461AY6361551EU4105091


EU4305461EU4105091


DQ0266691NR0411801


HQ4399051EU4305461


GQ3920581JF5025721


GU2657201AY999773




EF4942321NR0438221NR1126081NR1123971


99



GU2946851NR0255621NR0255611NR0255621NR0420401




FR8376311EU7411401JQ9244031

a

b bbc bc


bcdbcde

cdef

defef ef ef f f f f

0100200300400500600700800

BN3

BN24

BN4

BN82

BN25

BN13

BN69

GT1

5BN

48SB

31BN

17BN

73G

T39

GT6

GT1

BN71

BN23

BN68

SB30

BN12

BN9

GT2

BN2

BN72

SB22

BN22

BN7


(mg

of le

admiddotgminus1of

bio

mas

s)


























001





T Control


strain


strain











4





2







5 Conclusion






References



















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



ologicalcharacteris

ticso


lates

Characteris

tics

Strains

BN3

BN4

BN24

BN68

BN72

BN13

BN17

BN22

BN25

BN48

BN7

BN73

GT1

GT2

SB30

GT15

GT6

GT3

9BN

82BN

2BN

9BN

12BN

69BN

71SB

22BN

23SB

31ISP2

++++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

ISP4

+++

+++

++++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

++++

+++

+++

+++

+++

+ISP5

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

+++

Aria

lspo

remass

WG

Ow

Ow

GW

GG

GG

GV

PiW

CW

WW

WW

WW

WW

WW

CColon

yreverse

BT

YB

BB

TGr

TB

BP

BB

LbB

LbLb

BB

TT

TB

TB

LbSolublep

igment


Melanin

ontyrosin


Csource

utilizatio

nSucrose


+minus

+++minus

++

+minus

++++

+++

+minusminus

+minus

+++

+++minusminusminus

Fructose


+++

++++minusminus

++++

+++

+++

+++

+++

+++

+++


+++minusminus

Glucose

++

++

++

++

++

++

++

++

++

++

++

++

++

+++

Arabino


+++minus

++minus

+++

+++minus

+++

+++

+++minusminusminus

++

+minusminus

+Maltose


+++

+++

+minus

+++

+++

++++

+++

+minus

+++

+++


+++minusminus

Manno

seminusminus

++minus

+minusminus

+minusminusminus

+++

++++

++minus

+++

++++

+minusminusplusmn

+++

+minusminusminus

Mannitol

minusminus

++minus

+++

++++

+minus

+++minus

++++

+++

++

++++

+++


+++

+++minusminusminus

Galactose


+++

+plusmnminus

+++minus

+++

++++

+++

+++

+++


+++minusminus

+Inosito

lminusplusmn

++minusminusminus

+++minus

++++

++++

+++

++

+++

+++

+++minusminusminus

+++

+minusminusminus

+Rh

amno


+++


+minusminusminus

+Sorbito



+++

+minus

+++

+++

+++minusminus

++minus


Lactose


+minusminus

++

+++

+++

+++

++++

++minusminusminus

++++

+++

+++

++minusminus

Dextrin


+++

+++minus

++


++ldquo+rdquoT

estedpo

sitiveutilizedas

substrateldquominus

rdquotestednegativ

eno

tutilized


rdquolow

grow

th

Bbrow

nC

colorle

ssG

grayGrgreen

OwoffwhiteLblight

brow

nP

purpleP

ipink

TtanV

violetWw

hiteY

yello

w







AJ0020831DQ0266691NR 0411001


GU2134921NR0412091





GU2134921NR0410771




EU4305461AY6361551EU4105091


EU4305461EU4105091


DQ0266691NR0411801


HQ4399051EU4305461


GQ3920581JF5025721


GU2657201AY999773




EF4942321NR0438221NR1126081NR1123971


99



GU2946851NR0255621NR0255611NR0255621NR0420401




FR8376311EU7411401JQ9244031

a

b bbc bc


bcdbcde

cdef

defef ef ef f f f f

0100200300400500600700800

BN3

BN24

BN4

BN82

BN25

BN13

BN69

GT1

5BN

48SB

31BN

17BN

73G

T39

GT6

GT1

BN71

BN23

BN68

SB30

BN12

BN9

GT2

BN2

BN72

SB22

BN22

BN7


(mg

of le

admiddotgminus1of

bio

mas

s)


























001





T Control


strain


strain











4





2







5 Conclusion






References



















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology







AJ0020831DQ0266691NR 0411001


GU2134921NR0412091





GU2134921NR0410771




EU4305461AY6361551EU4105091


EU4305461EU4105091


DQ0266691NR0411801


HQ4399051EU4305461


GQ3920581JF5025721


GU2657201AY999773




EF4942321NR0438221NR1126081NR1123971


99



GU2946851NR0255621NR0255611NR0255621NR0420401




FR8376311EU7411401JQ9244031

a

b bbc bc


bcdbcde

cdef

defef ef ef f f f f

0100200300400500600700800

BN3

BN24

BN4

BN82

BN25

BN13

BN69

GT1

5BN

48SB

31BN

17BN

73G

T39

GT6

GT1

BN71

BN23

BN68

SB30

BN12

BN9

GT2

BN2

BN72

SB22

BN22

BN7


(mg

of le

admiddotgminus1of

bio

mas

s)


























001





T Control


strain


strain











4





2







5 Conclusion






References



















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology























001





T Control


strain


strain











4





2







5 Conclusion






References



















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


T Control


strain


strain











4





2







5 Conclusion






References



















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




2







5 Conclusion






References



















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





References



















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

research article resistance to and accumulation of heavy

Documents