Bacterial diversity in a soil sample collected from uranium mill-tailings called Gittersee and situated near the city of Dresden, Germany, was analysed by using a culture-independent 16S rDNA approach exploiting PCR ampli�cation primers 7F and 1513R. The results were compared with those obtained earlier analysing the same sample by using another primer pair, namely 43F-1404R [1]. The two 16S rDNA approaches demonstrated that Proteobacteria were the most predominant group in the sample, followed by Cytophaga/Flavobacterium/Bacteroides and by Gram positive

bacteria with low and with high G+C content, too. A large number of 16S rDNA sequences from two libraries were identical or almost identical. However, the ratio between the bacterial groups represented in them signifcantly differed.

7F-1513R primer set retrieved in addition to the above mentioned sequences also 16S rRNA genes, those of green non-sulphur bacteria and representatives of AD1 and OP11 divisions. The latter indicates that 7F-1513R primer set seems to be more reliable in analyses of bacterial diversity

Satchanska, Galina

Selenska-Pobell, Sonja

Italian

New Bulgarian University Scholar Electronic Repository

®ª« ¤¨ ­  º«£ °±ª ²   ª ¤¥¬¨¿ ­  ­ ³ª¨²¥Comptes rendus de l'Academie bulgare des SciencesTome 58, No 4, 2005BIOLOGIEMicrobiologieCULTURE-DEPENDENT APPROACH FOR DETERMININGMICROBIAL DIVERSITY IN SOILS FROM KCM/AGRIAREGION1G. Satchanska, I. Ivanova, V. Groudeva, E. N. Pentcheva,T. Kerestedjian, E. Golovinsky(Submitted on December 22, 2004)AbstractSignicant shifting of the microbial communities structure was detected in threemetal-polluted soils collected around the Pb-Zn smeltery KCM and the agrochemicalfactory AGRIA situated near the town of Plovdiv, Bulgaria. Industrial-, dumpsite-and agricultural soils were contaminated with As: 7.5-52.9 mg/kg; Hg: 0.086-0.404mg/kg; Cd: 2.3-71.1 mg/kg; Mn: 742-1510 mg/kg; Pb: 138-2560 mg/kg; Cu: 32-268 mg/kg and Zn: 293-4490 mg/kg. Remarkable ecological disturbance was foundin the agricultural soil using ecotoxicological test with type strain Pseudomonasputida DSM 50026 (ISO 10712). In order to assess ecologically relevant bacteriaover twenty soil bacterial isolates were cultured. Six of them possess tolerance toone or more heavy metals. Four of the isolates demonstrated herbicide tolerance to2,4-dichlorophenoxyacetic acid. Our results suggest that the microbial communityresponds to long-term metal- and pesticides contamination through changes in mi-crobial community structure and selection for resistance.Key words: polluted environments, microbial community structure, tolerantbacteria1Chemical analysis in this work was supported by grant NATO Science for Peace-973739.409Introduction. Pollution of the environment with heavy metals and organic com-pounds took a threatening size in the last decades. The harmful eect of heavy metalslike Pb, Cd, Mn, Cu, Zn is multiplied in cases when they interact with other pollutantslike xenobiotics [1]. Bacteria living in high metal concentrations develop abilities toresist such stress [2{4].Microorganisms growing in the presence of extreme metal concentrations possessspecic mechanisms of resistance as exclusion of metal ions, binding of metal ionsin the cell wall or intracellular sequestration in specic binding components as met-allothionein. Heavy-metal ions Hg2+, Cd2+, Ag+, Cu+, Zn2+and Pb2+form strongtoxic complexes in the cell which makes them dangerous for any physiological function[5, 6]. Dierent molecular and biochemical processes may cause adaptive response inmicrobial community exposed to heavy metals:(i) Induction of specic enzymes in the bacteria of microbial community resultingin an increase of degradative capacity of the total community;(ii) Selective growth of a specic subpopulation of a microbial community able totake up and metabolize the pollutant;(iii) Selection of mutants which possess specic metabolic capacities.Bacteria play a signicant role in migration and detoxication of a large num-ber of heavy metals due to bacterial ability to interact and biotransform them [7{9].Metal-microbe interactions have an important role in several biotechnological appli-cations including biomineralization, bioremediation, bioleaching and gained growingattention in recent years [10]. This development use indigenious isolates or microbialcommunities that are capable to mobilize and immobilize metal ions. The informationabout the diversity and activity of the indigenious microora in metal-polluted soils isof basic importance for understanding the biogeochemical processes occurring in theseenvironments. The problem of pollution with mixed wastes, especially environments co-contaminated with both metals and herbicides, requires isolation of pollutant-tolerantstrains [11].The aim of the present work was to derive information about the metal pollutionin soils around the Pb-Zn smeltery (KCM), Plovdiv, Bulgaria and to assess ecotoxi-cology eect of the pollutants. In addition to isolate and characterize environmentalbacteria and to test them for heavy metal and pesticide tolerance. This is the rstprofound investigation of the bacterial diversity in the co-contaminated area of theKCM smeltery.Materials and methods. Site history and sample collection. The investi-gations were carried out in the region with strong industrial activity in South Bulgaria.KCM-S.A. is the biggest Pb-Zn smeltery in Bulgaria and AGRIA is a factory for pro-ducing of agrochemicals for plant protection. KCM started in 1961 and now produce56000 t/y of zinc, 48000 t/y of lead, some precious metals and their alloys. Closelylocated AGRIA has output sheet containing over 40 herbicides, fungicides and insec-ticides. Both enterprises have been working for decades and cause co-contaminationwith heavy metals and pesticides of soils and waters in the area.Out of 66 soil monitoring points three highly metal-contaminated soil samples werechosen. Soil samples were collected in March 2002 under sterile conditions, transportedin ice to the laboratory and kept in refrigerator at 4C. The analyses started 24 h aftercollection.Sample 1 represents soil from industrial area, sample 2 { soil from dump site andsample 3 { soil from an abandoned agricultural eld.Metal-content analysis. Metal-content analysis was performed according toStandard Methods for Examination of Soils (EC). The value of the following elementsAg, Al, As, Ba, Be, Bi, Ca, Cd, Co, Cr, Cu, Fe, Ga, Hg, In, K, Li, Mg, Mo, Na, Ni,Pb, Se, Si, Sn, Sr, Te, Ti, Tl, U, V and Zn was investigated. The analyses were carriedout by HR-ICP-AES Jobin Yvon ULTIMA 2, in conformity with ISO 11885, after410dissolution of the solid phase with HNO3(also in exchangeable phase after extractionwith MgCl2).Microbial community structure analysis. Soil microbial counts were de-termined by the plate count method for viable cells. Soil suspensions were preparedby shaking 1 g soil in 9 ml sterile distilled water for 30 min. Serial dilutions from10 1to 10 7were surface spread on selective agar plates. CFU were counted after 2,5, and 7 days at 30 and 37C. The analysis includes the following microbial groups:heterotrophic aerobes, heterotrophic anaerobes, spore-forming bacteria, denitrifyingbacteria, amonifying bacteria, nitrifying bacteria, Fe(II) oxidizing bacteria, Mn(II) ox-idizing bacteria, Fe(III) reducing bacteria, Mn(IV) reducing bacteria, colourless sul-phur bacteria, cellulose degradating bacteria, oligocarbophiles, actinomycetes, fungi[12{18]. Additionally the presence of metal-leaching bacteria Acidithiobacillus thioox-idans, Acidithiobacillus ferrooxidans, Acidithiobacillus denitricans, Acidithiobacillusthioparus was investigated. All chemicals used in the analysis were supplied by Merckand Fluka (Germany). Nutrient media were supplied by Difco (USA).Isolation of novel bacterial cultures. Bacterial cultures were selected onthe basis of their morphology in nutrient agar and picked up from a single colony atthe end-point serial dilution. The isolation of microorganisms was performed usingmethods described elsewhere [19]. Heterotrophic aerobes (named R) and spore-formingbacteria (named RG ) were maintained at 4C and stored in glycerol at  20C forfurther investigation.Ecotoxicology test. The ecotoxicological test was carried out according tothe international standard method EU-ISO/DIS 10712.2: Pseudomonas putida growthinhibition test for determining the inhibitory eect on Pseudomonas putida DSM 50026(1995) [20]. 1 mL of mid-exponential phase P. putida was mixed with 5 mL of waterextract of each soil sample and 4 mL minimal medium and further cultivated for16 h/22C/180 rpm. In the control instead of contaminated water an equivalent volumeof mineral medium was added. Each sample was analysed spectrophotometrically at 450nm using Specol 11 (Carl Zeiss, Jena). Minimal medium contained for the preculture1g NaNO3, 0.24 g K2HPO4, 0.12 KH2PO4;0.1 g yeast extract, 10 g glucose, 0.4 gMgSO4;0.0001 g Fe citrat in 1000 mL dH2O, pH 7.0, and was autoclaved at 0.5 atmfor 20 min. For the test culture yeast extract and glucose were eliminated according toISO protocol.Tolerance to heavy metals. The novel bacterial cultures were tested for metaltolerance to the most dangerous for the environment and human health metallic cations.Stock solutions of 1 mM CuSO4, 1 mM ZnSO4, 4 mM Pb(NO3)2, 20 mM MnSO4and0.5 mM CdCl2were prepared in distilled water and sterilized for 20 min at 0.8 atm.Bacterial isolates were grown for 48 h at 30C in the presence of above mentionedmetals. Minimal media contained per litre 1 g NaNO3, 0.24 g K2HPO4, 0.12 KH2PO4,0.1 g yeast extract, 10g glucose, 0.4 g MgSO4, 0.0001 g Fe citrat.Tolerance to 2,4-D. The isolated bacterial cultures were also tested for resis-tance to the chlorophenolic herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) too. 2,4-D(C8H6Cl2O3) is a poorly degradable and third worldwide distributed herbicide anddefoliant. Bacteria were grown in mineral medium containing per litre 1 g NaNO3,0.24 g K2HPO4, 0.12 KH2PO4, 0.1 g yeast extract, 10g glucose, 0.4 g MgSO4, 0.0001g Fe citrat, with 0.1 mM and 1 mM 2,4-D added. Bacterial isolates were grown in thepresence of 0.1 mM and 1 mM 2,4-D for 48 h at 30C.Results and discussion. The experimental data shown in Table 1 demonstratethat soils in the KCM area were strongly polluted with Mn, As, Cd, Zn, Cu and Pb.The amount of the remaining elements did not signicantly exceed the standards.The highest metal concentrations were registered in agricultural soil. In this soilthe content of Cd, Pb and Zn exceeded in times the maximum permissible limit (MPL).The possible reason for strong contamination of the sample is the dust emissions coming411T a b l e 1Content of trace elements in soils from KCM-AGRIA (mg/kg)Sample As Hg Cd Mn Pb Cu ZnIndustrial soil 19.3 0.310 21.4 1340 990 268 1810Dumpsite soil 7.5 0.086 2.3 742 138 32 293Agricultural soil 52.9 0.404 71.1 1510 2560 256 4490Bold numerals show the values exceeding maximum permission limitfrom the KCM chimney. The analysis of elements bound as exchange cations in soilphases shows that the most mobile and with potentially harmful eect element wasCd. Other elements presented in minor amounts as exchange cations were Cu, Zn andMn. Above mentioned heavy metals considered to be of concern { As, Mn, Cd, Zn, Cuand Pb could readily include in the food chain and further damage the human health.Fig. 1. Quantitative analysis of microbial communities in KCM-AGRIA soils412High metal contaminations in the studied area aected the microbial communityliving in the polluted area and especially acted some microbial groups. Heterotrophicbacteria which require organic supplement for growth and energy supply may con-tribute to metal leaching. The heterotrophic microorganisms do not have any benetfrom the metal leaching. Among the bacteria members of genus Bacillus and genusPseudomonas are the most eective in metal solubilization. The quantitative microbialanalyses showed a pronounced prevalence of heterotrophic aerobes in the investigatedsoils: 104  105cells/g for industrial and dumpsite soils and 107cells/g for agriculturalsoil.Spore-forming bacteria were 105  106cells/g in all three samples (Fig. 1). Thehigh number of the latest bacteria in all investigated soils undoubtedly determined thisenvironment as extreme one.Denitrifying, amonifying and nitrifying bacteria showed concentrations of 103 105cells/g but nitrifying bacteria absented in the dumpsite soil and both nitrifying- anddenitrifying bacteria absented in the agricultural soil. As those bacterial groups serveas indicator of metal contamination in the environment, the results correlate with thedata from ICP analysis which shows that the agricultural soil is the most polluted one.Remarkable is the presence of Mn(II)- and Fe(II)- oxidizing bacteria presented inall samples at concentration of 103  105cells/g.Much recent research on dissimilatory metal-reducing microorganisms has focusedon their role in bioremediating of contaminated environments. Those microorganismsconsume oxygen and develop anoxic conditions [8]. Fe(III) reducing microorganismswere reported to degrade organic pollutants within contaminated aquifers and can con-vert U(VI) to U(IV). Fe(III)- and Mn(IV) reducing bacteria lessen iron and manganeseto their bivalent forms. They were presented with 103cells/g in all soil samples.Sulphate-reducing bacteria (SRB) which decrease the sulphate to hydrogen sul-phide in strictly anaerobic conditions, were found between 102and 104cells/g. SRBenzymatically mediate the reductive precipitation of toxic metals including U(VI),Cr(VI), Tc(VI) and As(V).Colourless sulphur bacteria, typical aerobic bacteria, showed a concentration of104cells/g soil.Cellulose degrading bacteria and oligocarbophiles showed very poor presence of10  103cells/g.Actinomycetes were found in the industrial soil with 103cells/g but they wereabsent in other two samples.Fungi can remove both soluble and insoluble metal species from solution and arealso able to leach metal cations from solid wastes. Many of them can produce organicacids which can also solubilize complex metal cations. Fungi were also detected onlyin the industrial soil and were not found in dumpsite and agricultural soil.Actinomycetes fungi also appear as indicator for environmental pollution and theirabsence demonstrated a change in the microbial community structure in samples 2 and3 and generally that the ecological balance in these soils is damaged (Fig. 1).The most active in bioleaching bacteria, belonging to genus Acidithoibacillus,were subject of special investigation. These are Gram-negative, non-spore formingrods which grow under aerobic conditions. Most thiobacilli are chemolithoautotrophicspecies which use carbon dioxide from the atmosphere as their carbon source for syn-thesis of new cell material. The energy derives from the oxidation of reduced sulphurcompounds, including sulphides, elemental sulphur and thiosulphate as nal oxidationproduct { sulphate [7]. Acidithiobacillus thiooxidans, Aciditthiobacillus denitricans,Acidithiobacillus novelus and Acidithiobacillus thioparus were not registered in any413soil sample. This fact is regular because these bacteria prefer the acidic pH while thesoil samples from KCM had a neutral pH.The biorestoration of environments polluted with mixed wastes, especially co-contaminated with metals and pesticides, requires isolation of strains that are bothmetal-tolerant and have a herbicide degradable capability. A total of 20 novel indige-nious soil bacterial cultures were isolated. Results showed that the bacterial culturesR4, RG1, RG2, R7, RG5, RG6demonstrate tolerance to one or more heavy metals. (seeTable 2.)T a b l e 2Metal resistance of novel bacterial isolatesEnvironmental bacteria Soil sample Metal toleranceR411 mM CuS041 mM ZnS0420 mM MnS04RG11 20 mM MnS04RG21 0.5 mM CdCl2R73 1 mM CuS04RG531mM CuS044 mM Pb(N03)220 mM MnS04RG63 1 mM CuS04All novel bacteria were also screened for herbicide tolerance to 2,4-D. Resultsshown in Table 4 demonstrated that the bacterial isolates R1, R3, RG3and RG5possesstolerance to 0.1 mM 2,4- D.Ecotoxicological test showed that all three soil samples inhibited to a dierentextent the growth of type strain Pseudomonas putida DSM 50026. Soil from agriculturalsoil (sample 3) had the greatest inhibiting eect on the growth of Pseudomonas putida {82%, followed by dumpsite soil (sample 2) and industrial soil (sample 1) with inhibitionof 70% and 54%, respectively (see Fig. 2)Conclusions. The study is providing information about the extent of pollutionin soils near the biggest Bulgarian Pb-Zn smeltery KCM. The change in the micro-bial communities structure in the studied soil samples correlates with the ICP dataof metal content. The results demonstrated that the structure of microbial communi-ties was aected in all investigated soils. Six of the novel bacterial isolates: R4, RG1,RG2, R7, RG5, RG6showed tolerance to one or more heavy metals. Four of the newlydiscovered bacteria R1, R3, RG3, RG5demonstrated slight herbicide tolerance. Ourresults suggest that the microbial community responds to long-term metal- and pesti-cides contamination through changes in microbial community structure and selectionfor tolerance. Further experiments of microbial degradation of organic pollutants to-gether with metal cations are nessessary to clarify the inuence of heavy metals on thebiorestoration potential.414Fig. 2. Ecotoxicological test { ISO/DIS 10712.2REFERENCES[1] Rieger P.-G., H.-M. Meier, M. Gerle, U. Vogt, T. Groth, H.-J. Knack-muss. J. Biotechnology, 94, 2002, 101{123. [2] Nakajima A., T. Sakaguchi. Appl. Envi-ron. Biotechnol., 24, 1986, 59{64. [3] Gadd G. In: Rehm H. J. (ed.) Biotechnology. Veinheim,1988, 401{433. [4] Ehrlich H. Appl. Microbiol. Biotechnol., 48, 1997, 687{692. [5] NiesD. Ibid., 51, 1999, 730-750. [6] Moore C., T. Di Christina. In: Environmental Micro-biology Enciclopedia (Ed. Gabriel Bitton), John Wiley&Sons, Inc., 2002. [7] Groudev S.,V. Groudeva. Micriobiol. Rev., 11, 1993, 261{268 [8] Lovley D. R. Features, 68, 2002,No 5. [9] Selenska-Pobell S., G. Kampf, K. Flemming, G. Radeva, G. Satchanska.Antonie van Leewenhoek, 79, 2001, 146{161. [10] Joerger K., R. Joerger, E. Olsson,C.-G. Granqvist. Trends in Biotechnology, 19, 2001, 15{20. [11] Hill R., C. Ezeani, O.Amund. American Society of Microbiology; 103rd General Meeting, May, Washington, 2003.[12] Silverman M., D. Lundgren. Ann. Rev. Microbiol., 50, 1959, 753{789. [13] LuefE., T. Prey, C. Kubicek. Appl. Microb. Biotechnol., 34, 1991, 688{692. [14] Leduc L.,G. Ferroni, J. Trevors. World J. Microbiol. Biotechnol., 13, 1997, 453{455. [15] DojkaM., P. Hugenholtz, S. Haak, N. Pace. Appl. Environ. Microbiology, 64, 1998, 3869{3877.[16] Fortin D., M. Roy, J. Rioux, P. Thibault. FEMS Microbiol. Ecol., 33, 2000, 197{208.[17] Chang Y., A. Peacock, P. Long, J. Stephen, J. McKinley, S. Macnaughton, A.415Hussain, A. Saxton, D. White. Appl. Environ. Microbiol., 67, 2001, 3149{3160. [18] EllisR., P. Morgan, A. Weightman, J. Fry. Ibid., 69, 2003, 3223{3230. [19] Karavaiko G.,G. Rossi, A. Agate, S. Groudev, Z. Avakyan. Biogeotechnology of Metals. Manual. (Ed G.I. Karavaiko) Centre for International Projects GKNT, Moscow, 1988. [20] ISO/DIS 10712.2Water quality-Pseudomonas putida growth inhibition test (Pseudomonas cell multiplicationinhibition test), 1995.Institute of Molecular BiologyBulgarian Academy of Sciences1113 Soa, Bulgariae-mail: sacanska@obzor.bio21.bas.bgGeological InstituteBulgarian Academy of Sciences1113 Soa, BulgariaFaculty of BiologySt. Kl. Ohridski University of Soa1164 Soa, Bulgaria416

Bacterial Diversity in the Uranium Mill-Tailing Gittersee as Estimated via a 16S rDNA Approach

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