Molecular monitoring of the microbial dynamics occurring on historical limestone buildings during and after the< i> in situ application of different bio-consolidation …

Molecular monitoring of the microbial dynamics occurring on historical limestone buildings during and after the< i> in situ application of different bio-consolidation …
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  See discussions, stats, and author profiles for this publication at: Molecular monitoring of the microbialdynamics occurring on historical limestonebuildings during and after the in situ...  Article   in  Science of The Total Environment · September 2011 DOI: 10.1016/j.scitotenv.2011.08.063 · Source: PubMed CITATIONS 22 READS 106 5 authors , including: Some of the authors of this publication are also working on these related projects: Yeast hybrids   View projectJörg EttenauerDanube University Krems 64   PUBLICATIONS   147   CITATIONS   SEE PROFILE Guadalupe PinarUniversity of Natural Resources and Life Scie… 245   PUBLICATIONS   1,548   CITATIONS   SEE PROFILE Katja SterflingerUniversity of Natural Resources and Life Scie… 286   PUBLICATIONS   2,225   CITATIONS   SEE PROFILE Fadwa JroundiUniversity of Granada 57   PUBLICATIONS   222   CITATIONS   SEE PROFILE All content following this page was uploaded by Guadalupe Pinar on 18 June 2015. 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All in-text references underlined in blue are added to the srcinal documentand are linked to publications on ResearchGate, letting you access and read them immediately.  Molecular monitoring of the microbial dynamics occurring onhistorical limestone buildings during and after the  in situ application of different bio-consolidation treatments  Jörg Ettenauer  a , Guadalupe Piñar  a, ⁎ , Katja Ster fl inger  a , Maria Teresa Gonzalez-Muñoz  b , Fadwa Jroundi  b a InstituteofAppliedMicrobiology,DepartmentofBiotechnology,ViennaInstituteofBioTechnology(VIBT),UniversityofNaturalResourcesandLifeSciences,Muthgasse11,A-1190Vienna,Austria b Department of Microbiology, University of Granada, Fuentenueva s/n, 18071 Granada, Spain a b s t r a c ta r t i c l e i n f o  Article history: Received 31 March 2011Received in revised form 24 August 2011Accepted 25 August 2011Available online 22 September 2011 Keywords: Molecular short- and long-term monitoringMicrobial communityLimestone In situ  bio-consolidation treatmentPCR-DGGEClone sequencing Microbially Induced Carbonate Precipitation is proposed as an environmentally friendly method to protectdecayed ornamental stone and introduced in the  fi eld of preservation of Cultural Heritage. Recent conserva-tion studies performed under laboratory conditions on non-sterile calcarenite stones have successfullyreported on the application of a suitable nutritional solution, inoculated and non-inoculated with  Myxococcus xanthus , as a bioconsolidation treatment. Furthermore, this procedure has been applied  in situ , very recently,to selected historical buildings in Granada, Spain. For the  fi rst time, we evaluate the ef  fi ciency and risks of the  in situ  application of the above mentioned treatments onto two historical buildings in Granada. The eval-uation consists of a detailed investigation of the micro-biota actively growing during the seven days of thetreatments  –  short-term monitoring and of that remaining on the stones after six and twelve months of theapplication  –  long-term monitoring. A molecular strategy, including DNA extraction, PCR ampli fi cation of 16S rRNA sequences, construction of clone libraries and  fi ngerprinting by DGGE (Denaturing GradientGel Electrophoresis) analysis followed by sequencing was used to gain insight into the microbial diversitypresent on the differentially treated stones. The monitoring of   M. xanthus  was performed by PCR usingspecies-speci fi c primers. Similar dynamics were triggered on both buildings by the application of the nu-tritional solution (inoculated or non-inoculated). 16S rDNA sequencing revealed the dominant occurrenceof members belonging to the Firmicutes and Proteobacteria during the seven days of the treatment,whereas after one year the order Bacillales of the phylum Firmicutes was the predominantly detected mi-croorganisms.  M. xanthus  could be detected only during the seven days of the treatment. The treatmentsseem to activate no dangerous microorganisms and furthermore, to select the remainder of a homoge-neous group of carbonatogenic bacteria on the stones after a long period of time.© 2011 Elsevier B.V. All rights reserved. 1. Introduction Microbially Induced Carbonate Precipitation (MICP) has been pro-posed as an environmentally friendly method of protecting decayedornamental stone. This method can be applied as a process of bio-deposition, i.e. the deposition of a protective surface layer withconsolidation properties as well as a process of bio-cementation,i.e. the generation of a biologically-induced binder (De Muyncket al., 2010).There are two main approaches regarding bio-deposition processes,those employing the use of calcinogenic microorganisms on stone sur-faces and those where no microorganisms are applied to the surface.Inthe fi rstseriesofapproaches,differentmicroorganismsandmetabolicpathways have been used for the precipitation of calcium carbonate(Adolphe et al., 1990; Castanier et al., 1999; Dick et al., 2006a; May, 2005; Rodriguez-Navarro et al., 2003). In the second series of ap-proaches, there are studies in which inducing macromolecules are sup-plied to the stone together with a supersaturated solution of calciumcarbonate(Tianoetal.,1999,2006).Inaddition,otherstudiesshowcar- bonateprecipitationbythemicro-biotainhabitingthestonebythemereaddition of an activator medium to the stone (González-Muñoz et al.,2008; González-Muñoz, 2008; Jimenez-Lopez et al., 2007, 2008). AlthoughMICPhasbeenwidelyinvestigatedunderlaboratorycondi-tions,furthertestsarenecessarytoprovetheviabilityandef  fi cacyofthisbacterial capability  in situ  under non-sterile conditions. This fact canconsequently affect the microbial activity of the inoculated carbonato-genicbacteriaaswellastheactivityofthosemicroorganismsinhabitingthe stone. To date, only a few published studies have implemented the in situ  application of MICP as consolidation treatment and haveaddressed the question of how the consolidation treatment is affectingthe micro-biota inhabiting the stone during and after the consolidation Science of the Total Environment 409 (2011) 5337 – 5352 ⁎  Corresponding author. Tel.: +43 1 47654 6943; fax: +43 1 3697615. E-mail addresses: (J. Ettenauer), (G. Piñar), Katja.Ster fl (K. Ster fl inger), (M.T. Gonzalez-Muñoz), (F. Jroundi). 0048-9697/$  –  see front matter © 2011 Elsevier B.V. All rights reserved.doi:10.1016/j.scitotenv.2011.08.063 Contents lists available at SciVerse ScienceDirect Science of the Total Environment  journal homepage:  treatment (Castanier et al., 2000; Jimenez-Lopez et al., 2007, 2008;  Jroundi et al., 2010; Le Métayer-Levrel et al., 1999; Orial, 2000; Piñaret al., 2010).As mentioned in the review of  De Muynck et al. (2010), the satis-factory application of MICP as a consolidation treatment by conserva-tors requires further knowledge not only of the effectiveness of themethod but also of the risk factors, especially the long term effectsoftheinoculatedbacteriaandtheappliednutrientmedia.In ouropin-ion, this additional evaluation parameter should be a pre-requisite toevaluating the effectiveness and risks of the treatments.In the present study we have evaluated the effect on the microbialpopulation inhabiting the stone during (short-term) and after (long-term) the  in situ  application of two different consolidation treatments.Theappliedtreatments,namelytheapplicationofasterilenutritionalso-lution inoculated and non-inoculated with  Myxococcus xanthus , have al-ready been tested for their ef  fi cacy in the consolidation of ornamentalstones under laboratory conditions ( Jimenez-Lopez et al., 2007, 2008;Rodriguez-Navarro et al., 2003). Recently, this optimized consolidationtreatment was applied, in a pilot-study, to two historical buildings  insitu . The two locations selected were the Monastery of San Jerónimo (SJ;Diego de Siloé, XVI Century) ( Jroundi et al., 2010) and the Royal Hospital(RH;EnriqueEgas,XVICentury).Bothbuildingsarebuiltofbuff-colouredbioclastic calcarenite stone extracted from the same quarry (Santa Pudiaquarries, Escúzar, Granada), and located in the same city (Granada,Spain) and, therefore, subject to the same climatic conditions. In the fi rst case, the authors performed enrichment cultures during the sevendaysoftreatmentandanalyzedthedynamicsofthemicrobialcommuni-ties by culture-dependent techniques ( Jroundi et al., 2010).Theresultspresentedin thisstudyarepart of this fi rst in situ  pilot-study. With the samples obtained we conducted short-term monitor-ing, over the period corresponding to the sevendaysof the treatment,in addition to the long-term monitoring of the bacterial communitiesinhabiting the stones, using a molecular strategy, performed six andtwelve months after the treatment, respectively.The goals of the present study were to investigate and compare a)the microbial communities inhabiting ornamental limestone fromtwo different buildings prior to any consolidation treatment, b) thedynamics of the community structure during the seven days of theapplication of two consolidation and one control treatments (short-term monitoring), c) the impact of the treatments on the inhabitingmicro-biota after six months and one year (long-term monitoring)and d) the prevalence of the inoculated bacterium,  M. xanthus , onboth buildings over the period of one year.The molecular strategy applied combined the  fi ngerprinting byDenaturing Gradient Gel Electrophoresis (DGGE), the constructionand screening of 16S rDNA clone libraries by DGGE and the sequenc-ing of selected clones. Furthermore, species-speci fi c primers wereused for the PCR detection of the inoculated strain  M. xanthus . 2. Materials and methods  2.1. Experimental procedure In order to analyse the micro-biota inhabiting the non-treatedstones of the two buildings under investigation, stone sampleswere collected from three selected sectors of each building prior tothe application of any treatment. Thereafter, the three selectedareas on the buildings each received a different treatment: (i) sterilewater as a control; (ii) a  M. xanthus -inoculated culture, and (iii) asterile M-3P nutritional solution. Thereafter, to evaluate the impactof these conservation treatments on the microbial community inha-biting the stones, two strategies were applied. Firstly: short-termmonitoring to identify the members of the microbial communityable to be activated quickly by the different treatments and, there-fore, potentially responsible for the production of calcium carbonatewithin the  fi rst week. To this end, enrichment cultures wereperformed on the 1st and 7th days of the application of the treat-ment. This monitoring was carried out by culture-dependent ( Jroundiet al., 2010) and  – independent techniques (this study).Secondly:long-termmolecularmonitoring(6 and12 monthsafterthe treatments) was performed to identify the fraction of the micro-bial communities remaining on the stones after this period of time.The bacteria identi fi ed during and after the treatments were com-pared with those inhabiting the non-treated stones. This monitoringwas performed by molecular means.  2.2. Stone-consolidation treatment  The decayed porous limestone (calcarenite) building blocks se-lected for the consolidation treatments in this study are part of an up-right wall exposed for centuries to outdoor weathering in the churchof San Jerónimo Monastery, and the Royal Hospital (Granada, Spain).In the case of SJ, the area selected for treatment has a southeast orien-tation and is located about 20 m above ground level. Treated sectorson the RH are located about 2 m above ground level on an outerwall with adjacent pavement (see Figs. 1 and 2).Areas were delineated in three sectors and the treatment on bothbuildings was carried out as described in Jroundi et al. (2010). Brie fl y,sectors A (SJ) and D (RH) (the control areas) were treated with steriledistilledwater;sectorsB(SJ)andE(RH)receivedacultureof  M.xanthus the  fi rst day of treatment and a sterile nutritional solution M-3P [1%Bacto Casitone, 1% Ca(CH 3 COO) 2 ·4 H 2 O, 0.2% K 2 CO 3 ·1/2 H 2 O in a10mM phosphate buffer, pH 8] (Rodriguez-Navarro et al., 2003) theremaining days of treatment; and sectors C (SJ) and F (RH) were onlytreated with the sterile nutritional solution M-3P. The treatment appli-cationwasrepeatedtwiceeverydayoverthesevendaysoftreatmenttokeep the stone adequately damp.  2.3. Sampling  Samplingwascarried outby twodifferentmethods:1)takingstonegrains directly from the surface of the stone before treatment and1 year after (in the case of San Jerónimo, samples were also taken6 months after treatment) and 2) applying  fi lter paper pieces to thestoneonthe fi rstandlastdaysoftreatments,asdescribedbelow,toob-tain bacterial pellets.  2.3.1. Stone sampling  Stone grains (about 300 mg) were taken from 3 different spots of each sector prior to the application of any treatment (hereafter re-ferred to as  ‘ non-treated stone ’ ) and after the treatments (at thetimesindicated above).These grainswereaddedto 1 mlof steriledis-tilled water, mixed, and aliquots were taken to perform serial dilu-tions and inoculations of different selective media. The remainingwater together with the stone grains was dried at 37 °C in steriletubes sealed with a 45 μ  m pore size sterile  fi lter. Dried stone grainswere frozen at − 80 °C for molecular analysis.The three stone samples corresponding to each individual sectorwere pooled prior to DNA extraction. In the case of SJ samples theywere called SJ-NT (non-treated stone), SA6, SB6, and SC6 for samplesafter 6 months of treatment, and SB12 and SC12 for samples after oneyear(seeFig.3).RegardingtheRoyalHospitalsampleRH-NT(non-trea-tedstone)wastakenbeforethetreatment;samplesSD12-1andSD12-2(sectorD),sampleSE12(sectorE)andsamplesSF12-1andSF12-2(sec-torF)were collected one yearafterthe consolidationtreatment(Fig. 4).  2.3.2. Sampling to obtain bacterial pellets Sterile and absorbent  fi lter paper (AFP, ANOIA, Spain) pieces (ca.1×2 cm in size), that can absorb a volume of 15 μ  l/cm 2 of solution,were used to collect the bacteria from the stone ( Jroundi et al.,2010). As soon as the treated areas were humidi fi ed on the  fi rst dayand just after the last application (day 7) of treatment, the paper 5338  J. Ettenauer et al. / Science of the Total Environment 409 (2011) 5337  – 5352  pieces were placed for 5 minutes on several spots (from 1 to 9 and 19to 27, see Figs. 1B and 2B) on the surface of the stone blocks. Each asepticallycollectedAFPpiecewasimmediatelyaddedto1 mlofster-ile M-3P nutritional solution and transported to the laboratory forcultivation analysis. The tubes with the absorbent paper strips weresubsequently made up to 5 ml with M-3P culture medium and incu-bated for 24 h. After this incubation time, the enrichment cultureswere centrifuged (at 10,000×  g   for 10 min) and the pellets were fro-zen ( − 80 °C) for further molecular analysis.  2.4. DNA extraction and PCR analyses DNA from stone slabs as well as from the collected bacterialpellets was extracted according to the protocol described bySchabereiter-Gurtner et al. (2001).Forall PCRreactions2×PCRMasterMixfromPromega[50units/mlof TaqDNA Polymerase in a supplied reaction buffer (pH 8.5), 400 μ  MdATP, 400 μ  M dGTP, 400 μ  M dCTP, 400 μ  M dTTP, 3 mM MgCl 2 ] was di-luted to 1x and 50 pmol/ μ  l of each primer was applied to the reactionvolumes. Two PCR reactions were performed to amplify eubacterial16S rDNA fragments for genetic  fi ngerprinting by DGGE. First, primers341f (Muyzer et al., 1993) and 907r (Teske et al., 1996) were used for the DNA ampli fi cation. Second, a semi-nested PCR using primers341GC and 518r (Neefs et al., 1990) was performed for DGGE analyses.PCRproductswerepooled,precipitatedinethanoland20 μ  lwasloadedontotheDGGEgel.AllPCRreactionswereperformedinanMJResearchPTC-200 Peltier Thermal Cycler with the thermocycling programs de-scribed bySchabereiter-Gurtner etal., 2001. Seven μ  l ofeachPCRprod-uctwasrunona2%(w/v)agarosegelforabout35 minat110 V,stainedin an ethidium bromide solution [1 μ  g/ml; stock: 10 mg/ml] for 15 – 25min and visualized by a UVP documentation system (BioRad Trans-illuminator, Universal Hood;Mitsubishi P93D-printer).A negative con-trol wascarriedout inall PCRreactions, wherenotemplate was added,to exclude the possibility of cross-contamination.  2.5. FingerprintanalysisbyDGGE  — DenaturingGradientGelElectrophoresis For DGGE  fi ngerprinting of the bacterial communities, the pooledPCR products supplemented with Loading Dye Solution (Fermentas),were run on gels in 0.5x TAE buffer [20 mM Tris, 10 mM acetate,0.5 mM Na 2 EDTA; pH 8.0] for 3.5 hours at 200 V and 60 °C in aBIORAD-DCODE ™ —  Universal Mutation Detection System (Muyzer etal., 1993). Fig.1. Treatedstone sectorsof MonasteryofSan Jerónimo, Granada (Spain):A. Wall areashowingthethree sectors selectedfor the treatments with:A,waterasa control;B, nutritionalsolutioninoculatedwith M.xanthus the fi rstdayandwithM-3Pculturemediumtherestofthedays;C,liquidM-3Pculturemedium.B.Schematicrepresentationofthesectorsusedforthetreatments, indicating the sizes of the three areas. The circles and numbers 1 to 9 and 19 to 27 correspond to the sampling points for culture-dependent studies.5339  J. Ettenauer et al. / Science of the Total Environment 409 (2011) 5337  – 5352  A chemical gradient ranging from 30 to 55% of urea andformamide in an 8% (w/v) polyacrylamide gel (BioRad, Munich,Germany) was used to separate the DNA bands from the differ-ent bacterial members. Staining of the denaturant polyacryl-amide gels was done in a 1  μ  g/ml ethidium bromide solution[stock: 10 mg/ml] for 15 – 25 min and afterwards visualized bya UVP documentation system (BioRad Transilluminator, Univer-sal Hood; Mitsubishi P93D-printer).  2.6. Construction of 16S rDNA clone libraries For the construction of clone libraries 2×3 μ  l DNA templates of eachsample were ampli fi ed in 2×50 μ  l reaction volumes using the universalprimers 341f and 907r. Aliquots of PCR products were analysed on a 2%agarose gel and further puri fi ed using the QIAquick PCR Puri fi cation Kit(Qiagen), following the protocol of the manufacturer. The puri fi ed DNAwas again visualized on a 2% agarose gel.Avolumeof5.5 μ  lofthepuri fi edPCRproductwasusedasaligationtem-plateforthepGEM — TeasyVectorsystem(Promega)followingtheman-ufacturer's instructions. One shot TOP10 cells (Invitrogen) were used forthetransformationreactions.Thesecellsallowtheidenti fi cationofrecom-binants(whitecolonies)onanindicatorLBmediumcontainingampicilline(100 μ  g/ml), streptomycine (25 μ  g/ml) and X-Gal (5-bromo-4-chloro-3-indolyl-ß- 1 -galactopyranoside;0.1mM)(Sambrooketal.,1989).  2.7. Screening of the bacterial clones About 50 to 150 white clones from each clone library were har-vested, and screened on DGGE as described by Schabereiter-Gurtneret al. (2001). The band positions of the bacterial clones were Fig. 2.  Treated stone sectors of the Royal Hospital, Granada (Spain): A. Wall area showing the three sectors selected for the treatments with: D (divided into the subsectors D-1 andD-2), water as a control; E, inoculated with  M. xanthus  the fi rst day and with M-3P culture medium the rest of the days; F (also divided into the subsectors F-1 and F-2), liquid M-3Pculture medium. B. Schematic representation of the sectors used for the treatments, indicating the sizes of the three areas, symbols as for Fig. 1.5340  J. Ettenauer et al. / Science of the Total Environment 409 (2011) 5337  – 5352
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