Chemical and Microbiological Characterization of Atmospheric Particulate Matter during an Intense African Dust Event in Southern Spain

Chemical and Microbiological Characterization of AtmosphericParticulate Matter during an Intense African Dust Eventin Southern SpainAna Sanchez de la Campa,†,‡,∥ Adela García-Salamanca,‡,∥ Jennifer Solano,§ Jesus de la Rosa,†

and Juan-Luis Ramos*,†,‡

†CIQSO- Associate Unit University of Huelva “Atmospheric Pollution”, University of Huelva, Campus El Carmen,E-21071 Huelva, Spain‡Estacion Experimental del Zaidín, CSIC, C/Profesor Albareda 1, E-18008 Granada, Spain§Bio-Iliberis R&D, Poligono Juncaril, Calle Capileira 7, 18210 Peligros, Granada, Spain

*S Supporting Information

ABSTRACT: This study presents the results of the physicochemicalcharacterization of particulate matter associated with an important dustevent from the Sahara area that occurred in the South of Spain in 2010. Thechemical composition of the samples reflected the dominance of the crustalcomponent of sand from the Sahara desert, although the presence of Mo, Ti,and V trace elements indicated that the dust contained industrial material; pro-bably collected in its transport from Africa. Microbial biodiversity associatedwith the dust was low, but dominated by Firmicutes and Proteobacteria. SomeFirmicutes (belonging to the genus Bacillus and Sporosarcina) were cultured onsolid and liquid medium, which suggested that the transported microbes werealive or present as spores that germinated under favorable conditions. Thesecultivable microbes in the form of spores were highly resistant to desiccation,heat, and UV light.

■ INTRODUCTION

The Sahara-Sahel desert is one of the major sources ofwindblown dust in the Northern Hemisphere1,2 and 20% ofthis dust is estimated to have the right size for long-rangetransport.3 Increased desertification from natural processes andhuman agricultural practices (e.g., overgrazing),4 in conjunctionwith global climate changes is leading to the concomitantintensification of atmospheric dust loading events. Annual dustemissions from the Sahara-Sahel desert currently represent about1 billion tons.5,6 The direction of dominant winds and theatmospheric conditions influence the place to which the dustfrom North Africa is transported, and long distance transport ofdust to Southern and Central Europe has been described.7−9

Dust particles from the North of Africa can travel as far as theAtlantic islands,10−13 and even across the Atlantic Ocean,reaching North America, South America, and Barbados.14,15

The Sahara-Bodele depression in Africa has been proposed tobe the major source of dust entering the Iberian Peninsula fromNorth Africa, with a dominant strip covering Mauritania, Mali,and Southern Algeria.8,16,17 These dust events often affect theSouth of Spain because of the proximal geographical location, butthis dust can reach the Pyrenees and even penetrate into France.African dust has a significant effect on particulate matter of con-centrations around 10 μm (PM10) in the Iberian Peninsula,

17−19

and since these air masses are loaded with mineral dust particles,they modulate aerosol climatology in the Iberian Peninsula.18,19

The source of atmospheric dust is traced using models basedon the calculation of back trajectories and satellite imagestudies.16 The mineralogical and chemical composition of NorthAfrican aerosol has been studied by numerous researchers.Calcite (CaCO3), dolomite (CaMgCO3), quartz (SiO2), clayminerals (kaolinite, illite, smectite, palygorskite, and chlorite)and feldspars were found to be characteristic of Africanaerosol.20,21 Iron oxides and calcium sulfate have also beenfound to be associated with these dust particles.22,23 Reche et al.24

assessed the effects of Saharan dust inputs of particulate matter,total phosphorus, total nitrogen, and water-soluble organiccarbon on bacterial abundance in two alpine lakes in theMediterranean basin. However, the microorganisms associatedwith these African air masses that reach the Iberian Peninsulawere overlooked. The aim of this study was to characterizeairborne microorganisms during an African dust storm reachingthe South of Spain. To this end we used a high-volume samplerequipped with TSP (total suspended particles <30 μm,aerodynamic diameter) inlets. Biodiversity associated with dustwas estimated using 16S rDNA analysis upon DNA extractionfrom air filter samples, and amplification/cloning; an effective

Received: December 18, 2012Revised: March 11, 2013Accepted: March 13, 2013Published: March 13, 2013

Article

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approach for identifying microbes present in different samplescollected from different environments.25−29 Our results showthat there are microorganisms that exhibit high survival uponexposure to UV light, heat, and desiccation conditions associatedwith dust from the Sahara-Sahel desert.

■ MATERIALS AND METHODS

Sample Collection and Chemical Analyses of Partic-ulateMatter.The sampling point was a rural monitoring station(CIECEM); 37° 0.98′ N, 6° 34.18′ W) located near DonanaNational Park in the southwestern corner of Spain. The

Table 1. Major (μg m−3) and Trace (ng m−3) Element Analyses Determined by ICP-AES and ICP-MS, Respectivelya

date start 18/03/2010 19/03/2010 20/03/2010

date end 19/03/2010 20/03/2010 20/03/2010

time start 10:00 10:00 10:25

time end 10:00 10:00 22:25

volume (m3) 959.4 959.3 479 mean concentration during nondust period (year 2010)

concentration (μg m−3) 103 162 165 26.0Ctotal 3.55 5.70 6.69 2.51CO3

2− 10.8 21.0 23.7 1.69SiO2 19.4 50.3 78.6 1.20Al2O3 6.48 16.8 26.2 0.43Ca 4.08 8.47 11.0 0.70K 1.37 2.68 2.96 0.24Na 6.61 8.40 2.68 1.95Mg 1.87 3.32 2.87 0.32Fe 2.07 4.51 5.18 0.14PO4

3− 0.23 0.42 0.52 0.05SO4

2− 7.19 7.90 4.93 2.55SO4

2− anthropogenic 5.53 5.80 4.25 2.08SO4

2− marine 1.65 2.10 0.67 0.49NO3

− 7.76 7.34 5.24 1.39Cl 9.16 11.1 2.19 2.33NH4

+ 0.77 0.13 0.11 0.33sample code G1679 G1680 G1682

date start 18/03/2010 19/03/2010 20/03/2010

date end 19/03/2010 20/03/2010 20/03/2010

time start 10:00 10:00 10:25

time end 10:00 10:00 22:25

volume (m3) 959.4 959.3 479 mean concentration during nondust period (year 2010)

concentration (ng m−3)Li 0.96 5.80 7.51 0.14Sc 0.29 1.51 1.80 0.02Ti 204 462 561 15.4V 9.96 31.3 19.9 3.55Mn 37.0 82.9 97.8 3.20Co 0.49 2.28 2.36 0.21Ni 1.36 13.8 12.3 1.52As 0.41 1.61 2.13 1.01Se 0.43 1.24 3.06 0.40Rb 2.37 10.4 11.2 0.36Sr 10.25 46.7 57.3 2.12Nb 0.32 1.53 1.65 0.04Cd 0.07 0.11 0.15 0.24Sn 0.28 1.17 4.22 0.74Sb 0.35 0.86 1.18 0.27Cs 0.02 0.35 0.12 0.02La 1.26 5.80 5.78 0.19Ce 2.04 9.72 10.7 0.23Pr 0.09 0.82 0.51 0.03Nd 0.77 3.87 4.27 0.10Sm 0.16 0.76 0.76 0.02Pb 0.69 11.03 9.12 3.79Th 0.12 1.39 1.52 0.05

aMean concentration for the year 2010 during non-dust period has been summarized.

Environmental Science & Technology Article

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instrument used for sampling particulate matter was a high-volume sampler (CAVF-PM1025 model, 40 m3/h) manufac-tured by MCV S.A, Barcelona, Spain, equipped with MCV TSPinlets. Munktell quartz filters were used for sampling during astrong Saharan dust storm from March 18 to March 20, 2010.One 24-h sample was collected forMarch 18 and 19, and anotherfor approximately 10 h March 20, which matched the time frameover which the sand storm affected southern Spain the third day.Details of the sampling are summarized in Table 1. The chemicalanalyses from the daily sampling filters of aerosols allowed us toidentify changes in the concentration and components ofparticulate matter. For chemical analysis, filters were placed ina desiccator box for 24 h at 20 °C and 50% relative humidity(RH), filters were then weighed with a Sartorius LA130 S-Fbalance (0.1 mg sensitivity).Once the mass levels of TSP were obtained, a portion of each

(75/400 cm2) was subjected to acid digestion (2.5mL:5mL:2.5mLHNO3/HF/HClO4) following the method proposed byQuerol et al.30 for the analysis of major and trace elements bymeans of inductively coupled plasma atomic emission spectrom-etry (ICP-AES) and inductively coupled plasma mass spectrom-etry (ICP-MS). The mean precision and the accuracy fell belowtypical analytical error and were in the range of 3−5% and <10%for ICP-AES and ICP-MS, respectively, and were controlled byrepeated analysis of 5 mg of NBS-1633b (fly ash) referencematerial (NIST, Gaithersburg, MD, USA). The detection limitsobtained were 0.01 ng/m3 for most of the trace elements weanalyzed. Blank filters were analyzed simultaneously from thebatches of their respective filter samples and the correspondingblank concentrations were subtracted from each sample.The soluble fraction of another portion of eachfilter (50/400 cm2)

was extracted with MilliQ-grade deionized water at 60 °C, andthe major ion content (SO4

2−, NO3−, Cl−, and NH4

+) in theleachates was determined by means of ion chromatographyDX-120 DIONEX with a separate column of anion IonPac AS9-HC 4 × 250 mm and suppressor membrane ASRS-ULTRA II(4 mm). A 9 mM sodium carbonate solution (suprarur quality)was used as eluent in an isocratic regimen to a flux of 1 mL/min.Finally, a circular section of each filter (250 mm in diameter) wasused to determine the total carbon (Ctotal) content using a LECOSC-144 DR instrument that measures CO2 resulting from theoxidation of the total C present in the sample. The lowerdetection limit was 0.1 μgC/m3.The content of silica and carbonate was indirectly determined

by stoichiometry from the concentrations of Ca, Mg, and Al, onthe basis of former experimental equations established by Querolet al.:30 3 × Al2O3 = SiO2; 1.5 × Ca + 2.5 × Mg = CO3

2−. Thelevels of Cnonmineral (or organic “OC” plus elemental carbon “EC”)were obtained by subtracting the Ccarbonate from the Ctotaldeterminations. Marine sulfate was indirectly determined bystoichiometry from the soluble Na+ levels from SO4

2−total/Na+

ratio in marine water (8.33 Na+ eq/Cl− eq).31

The size, morphology, and chemical composition of individualparticles was evaluated using an energy dispersive spectrometer(EDS, LINK-ISI model) mounted on a scanning electronmicroscope (JEOL-JSM5410 model). Conditions were set to15 kV accelerating voltage and 100 s of effective countingtime. Matrix corrections were made following the standardprocedures using a combination of silicate, oxides, and puremetals (wollastonite for Ca and Si, jadeite for Na, orthoclasefor K, corundum for Al, periclase for Mg, metallic Fe and Ti forFe and Ti).

Isolation of DNA from Filters, Construction of 16S RNAGene Libraries, and Sequencing. A piece (1/10 of the filter)from each of the three filters was cut and used for total DNAextraction with the commercial GNOMEDNA kit (QBIOgene)and total DNA was visualized by staining with ethidium bromidefollowing electrophoretic separation in a 0.8% (wt/vol) agarosegel. For PCR amplification of the bacterial 16S rRNA gene,universal primers F27 (5′-AGAGTTTGATCMTGGCTCAG-3′)and R1492 (5′-CGGYTACCTTGTTACGACTT-3′) wereused.32 DNA amplification was performed in a 25-μL reactionvolume with 2.5 U of recombinant Taq DNA polymerase, 25 ngof total DNA, 250 μM of each of the four deoxynucleosidetriphosphates, 1.5 mM ofMgCl2, 200 nM of each primer, and theappropriate buffer supplied by Roche. The PCR amplificationprogram was used as follows: one 5-min cycle at 95 °C; 30 cyclesfor 45 s at 95 °C, 45 s at 46 °C, 2min at 72 °C; and, finally, 1 cyclefor 10 min at 72 °C.PCR amplicons were purified by electrophoresis on 0.8%

(wt/vol) agarose gels. DNA was isolated from excised bandsusing a QIAQUICK Gel Extraction Kit (QIAGEN, Germany).The purified PCR products were ligated into plasmid vectorpGEM-T (Promega Corp., Madison, WI) with subsequenttransformation into competent E. coli (DH5α) cells. At least150 positive white clones were selected by blue and whitescreening. Bacterial rRNA clones were sequenced using primersM13 forward (5′-GACGTTGTAAAACGACGGCCAG-3′) andM13 reverse (5′-GAGGAAACAGCTATGACCATG-3′). TheGenBank accession numbers for the 16S rRNA gene sequencesanalyzed in this study are JQ622411 through JQ622531.

Phylogenetic Analysis of 16S rDNA Sequences. DNAwas sequenced using the Sanger Method in an ABI PRISM3730 × 1 capillary DNA analyzer. Sequences were manuallychecked to avoid ambiguities. Phylogenetic inference was carriedout using the ARB software package.32 Sequences wereautomatically aligned using SINA aligner against SILVA SSURef10033 and LTPs10034 reference alignments. Phylogeny wasreconstructed with the neighbor-joining algorithm using theJukes−Cantor correction for distance matrix calculation.

Index Calculations. Bacterial diversity was evaluated usingspecies-diversity indices.35 These pairwise distances served asinput to the DOTUR program for clustering tags intooperational taxonomic units (OTUs), generating rarefactioncurves and for calculating the Shannon−Weiner and Goodscoverage index.36−38 In addition, we determined the Chaononparametric index, as estimator of species richness.39−41

Isolation of Culturable Microorganisms from FiltersCollected after a Dust Storm. A piece of filter was suspendedin 1 mL of water and serial dilutions were spread on differentculture media solid agar plates; i.e. LB, 10-fold diluted LBmedium, andM9minimal mediumwith 5mMglucose, citrate, orbenzoate as a C-source.41 The total colony-forming units wereestimated to be in the range of 104 after 72 h incubation at 30 °C.

Characterization of Isolates. Spore Induction. Bacillussp. and Sporosarcina sp. were induced to sporulate by incubationin liquid Schaeffer’s Sporulation Medium (SSM)42 at 37 °C for48 h in a rotary shaker with vigorous aeration. Cultures wereharvested by centrifugation (10 000g, 10 min, 25 °C) and sporeswere purified using the lysozyme and buffer washing methoddescribed byNicholson and Setlow,43 heat-shocked (80 °C, 10min),and stored in deonized water at 4 °C.All spore preparations were determined by phase-contrast

microscopy to be essentially free of vegetative cells and to consistof >99% phase-bright spores. Their morphology was analyzed



using a LEO 1430VP scanning electron microscope. The viabletiter of spore preparations was determined by 10-fold serialdilution in phosphate-buffered saline solution and plating onSSM solidified with 1.7% (wt/vol) agar.Desiccation Assays with Vegetative Cells. Bacterial cells

were grown in Luria Broth (LB) at 30 °C overnight and adjustedto a cell density of ∼108 CFU/mL. Then 100-μL samples weredeposited into 96-multiwell plates. The plates were incubated ina chamber under controlled humidity (50−55%) and temper-ature (30 °C) and cells were allowed to dry. At t = 0 and whenindicated, cells from a well were removed and the viable oneswere counted on LB plates. At other time points distilled waterwas added to reach the original volume, and 30 min later viablecells were counted. The bacterial survival ratio is the ratio of thelog of the number of bacterial cells present in the suspension afterdesiccation divided by the log number of viable cells beforedesiccation multiplied by 100. These assays were also performed,as described above, but with 108 spores per mL.UV Resistance Assays with Vegetative Cells. Vegetative

cell cultures from 13 different isolates were adjusted to ∼1−2 ×108 CFU/mL. Cells were then diluted 10-fold (up to 108 fold)and 20 μL of each dilution was deposited on LB plates and left todry on the surface. Triplicate plates were then irradiated with UVlight (7 J/m2/s) for different time periods (0, 5, 10, 15, and20min). The irradiated plates were incubated in the dark for 24 hat 30 °C and the number of surviving cells was determined as

CFU of the dilutions yielding readable results. The same assayswere performed using spores instead of vegetative cells.

Heat Resistance Assay with Vegetative Cells. Vegetativecells from 13 different isolates grown at 30 °Cwere cultured up toa turbidity of approximately 1 at 660 nm. Different aliquots werethen prepared and incubated without agitation for 30 min at 30,37, 45, 55, 75, and 90 °C. The number of viable cells was thencounted by spreading serial dilutions in SSM medium. Theseassays were also performed as above but with 108 spores per mL.

■ RESULTS AND DISCUSSION

Aerosol Characterization.Total suspended particles <30 μm(TSP) were collected at the rural monitoring station mentionedabove during a dust storm from the Sahara desert over the IberianPeninsula March 18−20, 2010. Front maps of the Met Office ofU.K. (www.metoffice.gov.uk) and back trajectories indicated thatthe air mass during the event arrived at the Iberian Peninsulafrom the North African desert (Figure 1A and B show mapscorresponding to March 19). This Saharan air mass reached theSouth of Spain under a synoptic situation governed by thecombination of a low pressure center located at the West orSouthwest of the Iberian Peninsula and an anticyclone located atthe East/Southeast of the Iberian Peninsula (see isobar maps inFigure 1A). This scenario was previously described by Rodriguezet al.44 as one of synoptic pressure systems favoring the long-range transport of Saharan dust into the Iberian Peninsula.

Figure 1. Hysplit back trajectories of air masses arriving at the sampling station (A), isobar maps (B), and hourly levels of PM10, SO2, and NOx (C)during the North African dust storm.



www.metoffice.gov.uk

During this episode the hourly data registered by the monitoringstation for air quality in Andalusia revealed increased levels ofPM10, but not of NOx (sum of NO and NO2) and SO2 levels(Figure 1C), which confirms that the dust associated with the airmass was responsible for this increase. The air mass backtrajectories pointed toward the Saharan/Hoggar Massif area asthe origin of this episode. In this region local geology isdominated by carbonate depocenter caused by the opening ofthe Atlantic in the Triassic era, as well as by Precambrian igneousand metamorphic rocks from Western Sahara and the AlgerianHoggar Massif, respectively.45 Soils from the Hoggar Massifshow the greatest chemical diversity (e.g., abundant hornblendeand alkali feldspar, kaolinite, illite, montmorillonitic smectitewith minor calcite and paligorskite), whereas Western Saharansoils are rich in detrital carbonate grains (calcite and dolomite),and palygorskite-sepiolite mafic clays.45,46

The concentrations of collected TSP ranged from 103 (March18) to 165 μg m−3 (March 20). The results of ICP-AES andICP-MS data (Table 1) showed high concentrations of SiO2,CO3

2−, Cl, Na, Al2O3, Ca, NO3−, SO4

2−, and some trace elementssuch as Ti, V, Mn, Sr, Ba, Zn, Zr, Ni, La, Ce, and Pb duringSaharan dust input. The mean concentrations during non-Saharan dust periods (Atlantic air mass) have been included inTable 1, showing lower concentrations of Al2O3, Ca, K, Mg, Fe,SO4

2−, and NO3, and trace levels of Ti, V, Mn, Rb, and Srcompared to those obtained under the influence of Saharan dustinput. The high concentrations of the major elements identifiedreflected that the material collected in the TSP filters had thetypical chemical composition of particulate matter from thedesert, but that it was accompanied by trace elements that werenot characteristic of the area. Rodriguez et al.44 defined industrialemissions in North Africa and showed that the major source ofnitrate, sulfate, ammonium, Ti, V, and Mo was emissions fromcrude oil refineries, power plants, and phosphate-based fertilizerplants. Therefore, given the direction of the air masses and thecomposition of the TSP collected in Huelva, we propose that thedust collected there camemainly from the Sahara, although someadditional industrial emission material was picked up on its wayfrom the Sahara to Spain. To further confirm the source of theTSP, samples were analyzed by SEM-EDS (Figure 2). Particleswith irregular morphology and great size variability (<10−20 μm)composed mainly of Si, Al, K, Mg, Fe, Ca, ± Cl, ± S wereobserved (Figure 2a). Moreover, particles larger than >20 μm,made up of polymineral aggregates, were also present (Figure 2b).Other identified particles were Ca-rich particles that corre-sponded to calcium carbonate well-defined crystals smaller than5 μm (Figure 2c) and to particles with irregular morphologyassociated with Ca and Al (Figure 2c) particles located just to theright of the calcium carbonate crystal; Aulacoseira diatoms(Figure 2d) and sodium chloride that corresponded to well-defined crystals whose size was about 10 μm (Figure 2e),although noncrystallized sodium chloride was also present in thesamples. The presence of exotic particles of diatoms has also beenidentified in other studies.46,47

Microbes Associated with the Dust Intrusion. Theabove-reported episode represents the largest dust intrusionfrom the Sahara desert recorded in Spain in 2010, and possiblythe most important one in recent years. We determined thebiodiversity of prokaryotic microorganisms traveling in associ-ation with this dust by analyzing 16S rDNA sequences. To thisend total DNA from the filters was isolated and amplification of16S rDNA genes was performed using universal primers, asdescribed above.We obtained 121 sequences with an average size

of 1300 bp. Rarefaction analysis was carried out to estimatebacterial diversity, and the number of predicted bacterial OTUspresent in the air mass intrusion from North Africa was 19 whenthe OTU definition was set at a 3% distance level, 17 OTUs whenthe OTU definition was set at 5%, and 16 OTUs when the OTUdefinition was set at 10% (Figure 3). These data indicated a lowlevel of biodiversity associated with the collected dust particles.A series of statistical analyses were performed to estimate thedepth of the samples. The Chao 1 index suggested that themaximum expected OTU value for the dust event samples wouldbe 27, whereas the maximumOTU value in the rarefaction curvewas 19. Goods coverage index value was 0.85, and the Shannon−Weiner index value was 2.25, suggesting that the bacterialcommunity associated with the dust event is of low diversity andthat our analysis had sufficient depth.Most of the 16S rDNAs sequences obtained had >90%

similarity to numerous rDNA sequences deposited in GenBank.Microbes were identified at the phylum level. Themost abundantmicrobes associated with this dust event were Proteobacteria(74% of total) and Firmicutes (19% of total) with very minorpresence of Bacteriodetes and Actinobacteria (less than 1%), and5% sequences of unknown microbes. Within the phylumProteobacteria (Table 2) 16S rDNA sequences correspondedmainly to bacteria of the genus Erwinia (31.7%) andAcinetobacter(21.8%) (Table 2). Within the phylum Firmicutes 16S rDNAsequences defined organisms related to Lysinibacillus (14.6%)and Bacillus (4%) as the most abundant ones (Table 2). Theirlocation in the phylogenetic trees was based on the ARB program(see Supporting Information Figures S1 and S2). Other bacteriapresent in low proportions were identified as Rhizobiaceae, andSphingobacteriaceae.Polymenakou et al.47 examined the distribution of micro-

organisms associated with airborne particles over the eastern

Figure 2. SEM images showing morphology, composition, and grainsize of particles samples: (a), (b), (c) calcium carbonate crystal,(d) detail of an Aulacoseira diatom, and (e) sodium chloride crystal.



Mediterranean region during an African storm from the Saharaarea. As in our study, their 16S rDNA clone libraries were of lowdiversity, but, in their case, Firmicutes and clones affiliated withActinobacteria and Bacteriodetes were the most abundant. In along-range dust sample that traveled from Mali to the VirginIslands, Bacillus sp. were also described to be the most dominantmicrobes.49 Therefore, in all three studies in which dust from theSahara was collected in different regions the prevalence of spore-forming microorganisms is evident, suggesting their potentialcommon link to Saharan dust. The fact that the three studies alsoreported differences in the proportion and in the nature of theidentified microorganisms could be attributed to the fact thatsamples may originate from different sites in the Sahara or thatsome microbes were incorporated during dust transport to thedifferent locations in which the dust was collected.Analysis of Culturable Isolates. Griffin et al.48 and

Prospero et al.49 reported that viable (colony forming) bacteriawere recovered from African dust in samples collected in theCaribbean islands. Griffin et al.48 reported that in air samplescollected at St. John in the Virgin Islands during “African dust-events” Bacillus sp. constituted 38% of the bacterial isolates.Other frequently isolated genera were Kocuria (12.8%),Planococcus (8.5%), Saccharococcus (7.4%), and Micrococcus(6.4%). Air samples collected in the South of Spain during theMarch 2010 dust event were screened for the presence ofculturable microbes by plating serial dilutions on rich medium(LB), 1/10 LB medium, or M9 minimal medium with differentC-sources (see Materials and Methods). We selected thirteencolonies that weremacroscopically different, and their 16S rDNAgene sequences were determined. (The GenBank accessionnumbers for the isolates in this study are JQ622398−JQ622410).The analysis revealed that all microbes that grew on plates

belonged to the genus Bacillus, except for one that belonged toSporosarcina. Phylogenetic trees generated using ARB align-ments with these 16S rDNA sequences were reproducible andprovided consistent phylogenetic placements with the RDPtoolset (see SI Figure S2). Within the Bacillus genus, isolatesbelonging to the species Bacillus cereus, B. thurigiensis, B. safensisand B. megaterium were found. We should indicate that microbesother than Bacillus were viable in the samples (see Table 2),although the limited set of culture media used to recover themicrobes may have restricted the growth of these other microbes.Our data are consistent with those by Griffin et al.48 in the sensethat spore-forming and desiccation-resistant strains prevail indust samples from the Sahara, and these distinctive traits are mostprobably due to long-distance dust transport and bacterialsurvival. The fact that we recovered bacteria from two generainstead of five in the case of Prospero et al.49 or Griffin et al.48

may be related to the use of different culture media at thedifferent laboratories. The microorganisms that we measured inaerosols in Huelva are ubiquitous in soils and plants around theworld25,28 and since there is very little information on the soilsfrom the dust source regions in North Africa, the exact origincannot be determined. It should be noted that all isolates whenintroduced in planted soils were able to proliferate and reach celldensities in the range of 106 CFU per gram of rhizosphere soil.

Survival Mechanisms As a Means to Understand Long-Range Transportation. Prospero et al.49 and Griffin et al.48

suggested that viability in long-range transported microorgan-isms could be linked to resistance to desiccation, heat, and UVradiation. To further explore this hypothesis, we submittedvegetative cells and spores of the isolated microbes to heat, UVexposure, and desiccation and determined their survival overtime.

Figure 3. Rarefaction analysis of 16S rDNA sequence heterogeneity in clone libraries from samples collected during a Saharan dust event in SouthernSpain. The total number of screened clones is plotted against operational taxonomic units (OTUs) at 97% (○) and 95% (●), and 90% sequencesimilarity (△).

Table 2. 16S rDNA-Based Frequencies and Taxonomic Affiliation of the Sample Using ARB Classifier

phylum class family genus %a

Proteobacteria Gammaproteobacteria Enterobacteriaceae unclassified 12.5Erwinia 31.7Escherichia/Shigella 6.5

Moraxellaceae Acinetobacter 21.8Alphaproteobacteria Rhizobiaceae unclassified 1.6

Firmicutes Bacillaceae Bacillus 4Lysinibacillus 14.6

Bacteroidetes Sphingobacteriaceae unclassified 0.8Actinobacteria Nocardiaceae Rhodococcus 1.7

unclassified 5.6a% Means the portion of these microbes with respect to the total clones analyzed.



Ultraviolet resistance tests were performed with vegetativecells and spores from the 13 isolates. The results in Figure 4 arethe average of three replicates with a standard error below 10% ofthe given values. These thirteen isolates were grouped into threesets based on death kinetics of vegetative cells upon UVexposure. In group A we included isolates 1, 3, 5, 9, and 12characterized because they were the most sensitive to UV light(see Figure 4 for isolate number 1) and they were as resistant asthe control lab strain Bacillus subtilis. Strains in group C, isolates7, 11, 13, and 16, exhibited higher resistance to UV exposure withmaintenance of viable cells 10 min after exposure (see Figure 4for isolate 16). Group B included isolates 2, 4, 6, 8, 10, and 14 andexhibited an intermediate level of survival upon UV exposure(see Figure 4 for isolate 6). It should be noted that exposure ofspores from all 13 isolates to UV light revealed that they wereequally resistant and that the survival decay rate was lower thanthat of vegetative cells.When vegetative cells from the 13 isolates were submitted to

desiccation assays all of them exhibited relatively high tolerancewith at least 50% survival after 21 days of desiccation (Figure 5).Spores of lab B. subtilis strain and the 13 isolates exhibitedsimilarly high survival rates upon exposure to desiccationconditions (Figure 5).Vegetative cells from all 13 isolates also behaved similarly

regarding heat tolerance with a marked drop in survival at 45 °Cor higher, whereas the survival of the spores almost reached 100%at 55 °C and only started to decrease at 75 °C (Figure 6).

Since the main source of dust reaching the South of Spain isthe arid Sahara desert and since the culture media we used shouldhave permitted the growth of Proteobacteria and differentFirmicutes, but we only recovered Bacillus and Sporosarcina-spore forming Firmicutes as viable cells, we suggest that spores are

Figure 4. Ultraviolet resistance of vegetative cells of different isolates. The survival curves were obtained as indicated in Materials and Methods. Thesymbols used are: Bacillus subtilis as control (○), group A isolate 1 (●), group B isolate 6 (△), group C isolate 16 (▲). Viable cells were determined asdescribed in Materials and Methods.

Figure 5. Survival of spores from different isolates under desiccation conditions. The assay conditions are as described in Materials and Methods. Thesymbols used are: Bacillus subtilis as control (○), group A isolate 1 (●), group B isolate 6 (△), group C isolate 16 (▲).

Figure 6. Heat effects on Bacillus spp; spores and vegetative cells.Survival of vegetative cells (A) and spores (B) of different isolates todesiccation conditions. The assay conditions are as described inMaterials and Methods. The symbols used are: vegetative cells ofBacillus subtilis as control (○), group A isolate 1 (●), group B isolate6 (△), group C isolate 16 (▲), spores of Bacillus subtilis as control (□),group A spores isolate 1 (■), group B spores isolate 6 (◊), group Cspores isolate 16 (⧫).



the most likely form in which viable bacteria are transported fromthe Sahara desert to the South of Spain, although we cannotexclude that culture media bias the isolation of viable cells.Our results show that vegetative cells and spores of all isolates

were similarly resistant to heat and desiccation, although a widerange of sensitivities to UV light was observed. These resultssuggest that drought and heat tolerance could be commonnecessary traits to facilitate long-term transportation and survivalof microbes. During long-distance transport in the stratospheremicrobes are exposed to UV and since we had a wide range ofresistance to UV among our isolates, we suggest that dust couldactually protect cells from damage during transport. Althoughour study shows that bacteria are found in the presence of dust,we cannot determine whether the microorganisms are directlyassociated with individual dust particles or whether they areindependently suspended in the air.

■ ASSOCIATED CONTENT*S Supporting InformationFigures S1 and S2. This material is available free of charge via theInternet at http://pubs.acs.org.

■ AUTHOR INFORMATIONCorresponding Author*Phone: +34 958 181608; e-mail: [email protected];mail: C/Profesor Albareda 1, 18008 Granada, Spain.Author Contributions∥Authors A.S.C. and A.G-S. contributed equally to this work.

NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSWe gratefully acknowledge the NOAA Air Resources Laboratory(ARL) for the provision of the HYSPLIT transport model andMet Office for the provision of front maps. Work at ourlaboratories was supported by Fondo Social Europeo andFondos FEDER from the European Union and financiallysupported by project RNM2011-7800 of the AutonomousGovernment of Andalusia, and Projects CGL2011-28025 andCSD2007-00005 of the Spanish Ministry of Science andInnovation. We thank M.M. Fandila for secretarial assistance.

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Chemical and Microbiological Characterization of Atmospheric Particulate Matter during an Intense African Dust Event in Southern Spain