Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | ymetrix ff , 1 Discussions Biogeosciences , E. E. Smith 1 , S. Kim 1 1 6726 6725 , J. Gilberry 1 , and J. Quizon 1,2 er et al., 1997), studies of phylogenetic diversity using molecular ff , D. Cunningham 1,2 This discussion paper is/has been under review for the journal Biogeosciences (BG). Please refer to the corresponding final paper in BG if available. National Security Technology Department, The Johns Hopkins University Applied Physics Department of Civil and Environmental Engineering, University of Maryland, Baltimore Pinnick et al., 1995;Pan Reyes et et al., al., 2007; 1999; Steele et Lee al., et 2008). al., Several 2004; common Sivaprakasam observations et have arisen al., from 2004; including viruses, bacteria, fungi,imal plants (e.g. cells pollen or grains) cellambient and bacterial human fragments aerosols and (Matthias-Maser have other sought andsity, an- to variability Jaenicke, and provide factors 1995). a that better controlpast understanding Studies the studies of biological of the fall aerosol into diver- population. these oneteria In of and principal, three all fungi primary (Sha methods categories: such studies as clone of sequencing viable/culturablerays (Hua bac- et (Brodie al., et 2007; al., Neheme 2007), et al., or 2008) spectroscopic or characterization microar- of biological aerosol (e.g. aerosol. 1 Introduction Biological aerosol particles are thought to have a wide variety of biological sources, using culture and clinicalPhyloChips. biochemical tests, Comparing and the 16Seach culturable rRNA sample fraction diversity provides and using aallows surveying A comprehensive comparison the look total with at 16S previousparcels the rRNA studies. sampled, bacterial over of population the Thirty-six studiedmation two hour on day and the back-trajectories period sources of beginning ofApplied 4 aerosol the sampled November Physics air on 2008, Laboratory the provide campusmodeling in infor- of of Laurel, Johns air Hopkins MD. University parcels This may study provide indicates insights into that the back-trajectory observed diversity of biological Bacterial aerosol have beenthe observed mid and nineteenth century. studied These ining studies of the have sought the ambient to diversity, environment variability provideIn and a since this better factors understand- that study, we control show the comparisons biological between aerosol diversity population. of culturable bacteria and fungi, S. Ratnesar-Shumate 1 Laboratory, Laurel, MD, USA 2 County, Baltimore, MD, USA Received: 12 July 2010 – Accepted: 30Correspondence August to: 2010 J. – L. Published: Santarpia 3 ([email protected]) SeptemberPublished 2010 by Copernicus Publications on behalf of the European Geosciences Union. Abstract Biogeosciences Discuss., 7, 6725–6747, 2010 www.biogeosciences-discuss.net/7/6725/2010/ doi:10.5194/bgd-7-6725-2010 © Author(s) 2010. CC Attribution 3.0 License. Transport and characterization of ambient biological aerosol near Laurel, MD J. L. Santarpia 5 20 25 15 10 Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | C. The ◦ ect both the observ- ff ered saline (PBS). Four ff Actinobacteria, Firmicutes erm, 1995) and its diversity ff C for 24 h. SDA plates were separately ◦ 6728 6727 ects of precipitation. Collected aerosol samples were analyzed ff are ubiquitous in aerosol samples collected from diverse locations erential characteristics such as colony morphology, initial gram stain ff C and examined for growth every 24 h for up to 7 days. ◦ ciency values (Kesevan and Schepers, 2006), samples were collected ffi ymetrix PhyloChips (DeSantis et al., 2003). Using traditional culture and com- ff er et al., 1997; Brodie et al., 2007; Fierer et al., 2008; Mancinella and Shulls, ff Proteobacteria ymetrix, Santa Clara, CA), and another part was plated the same way as the pre- ymetrix PhyloChips (Fig. 1). ff All plates were examined for growth after an incubation period of 24 h at 37 Immediately after recovery from the OMNI, 200 uL of the aerosol sample was re- This study develops a method for understanding and comparing the molecular and ff incubated at 30 San Diego, CA) betweenusing sample a collections. laboratory PBS Negative solution,water control the used PBS experiments to that were replenish was run the used fluid to level collect in the the samples OMNI andresulting during the colony collection. forming unitslates based (CFUs) on were di groupedreaction, and and categorized results as ofbered basic unique and biochemical re-plated iso- on tests. TSA,plates, CA, Isolates other SBA, were MAC, than and then SDA, Sabouraud individually were Dextrose num- incubated Agar (SDA). at All 37 (A concentrated sample, whichthe allowed concentration for process, the servingevaluate comparison the as of viability a growth of concentrationretained. before cultured control and organisms. The and OMNI after helping Any was to remaining decontaminated further sample with was RNase labeled Away (Molecular and BioProducts, A moved and 50 uL aliquotsAgar were (CA), plated Sheep’s on Blood each10.8 mL Agar of of (SBA), Tryptic sample Soy and Agar wasTo MacConkey ensure (TSA), then Agar the Chocolate concentrated (MAC). free via The nucleiction centrifugation remaining acid process, at present 1 4500 in mL RPM thepellets. for of samples Part 10 the min. was of not the decanted concentrated lost supernatant sample in was was this analyzed used concentra- using to the PhyloChip resuspend GeneChip the sample collection e over shorter periods to examinetity any or short-term diversity. Due temporal to variationobservations changing in of weather bioaerosol conditions, the quan- this e alsoby allowed dividing some the anecdotal 11 mL sample for cultivation and biochemical tests and analysis using et al., 2007). Since the OMNI has a high sample collection rate and reportedly high and two from 13:30samples to used 17:30 EST in on PhyloChip 4 analysis and are 5 collected November over 2008. at least Typical environmental a 24 h period (Brodie ison with the broad range of previous studies. 2 Methods Air samples were collectedcyclone over (Evogen, a Inc., two Kansasimately day City, 277 Lpm MO) period into programmed using4-h approximately to an samples 11 collect OMNI mL were samples collected of 3000 during at phosphate wetted-wall the approx- bu sampling period, two from 05:30 to 09:30 EST November. After collection, aerosolusing samples A were characterized for bacterialmon diversity biochemical tests, theComparing diversity the of culturable fraction culturableprovides and a fungi comprehensive surveying and look the bacteria at total the was bacterial 16S examined. population rRNA studied of and each allows compar- sample (Sha 1978). Gram-positive speciesgram-negative dominate species are the the culturablebecause dominant fraction gram-negative fraction organisms of of tend to bioaerosols, the beaerosol nucleic while more collection acid fragile or and burden, even are existence probably less as likely an to aerosol. survive culturable prokaryotic diversity in thelected ambient for aerosol microbiological background. analysis Samples over were col- a two day period from 4 November through 5 their sources (Hua et al.,able 2007). biological Meteorological aerosol conditions concentration can(Brodie (Lighthart a et and al., Sha 2007; Fiererprints et have been al., found 2008). inand Diverse air bacteria samples and/or (Brodie their et al., genomic 2007). finger- these studies. Bacteria can be transported to geographically distant locations from 5 5 25 15 20 10 25 15 20 10 Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | was N where 2 N 13 = erential media types, and the isolates C. ff ◦ 80 − 6730 6729 Several controls were implemented to identify any false positive matches on the Phy- Once each sample was processed with a PhyloChip, the resulting data was analyzed Once the environmental samples were concentrated, the DNA was extracted using The PhyloChip microarray (DeSantis et al., 2003) used in this project takes advan- The range of media and protocols/procedures used in this study were limited by both After the re-plating and 24 h incubation period described above, isolates were further der packs arriving directly from the manufacturer, steps to eliminate this background and sample processing fluids.collection fluid As in part the theseevaporation OMNI controls, and during the the sample PBS water collection that aliquotsBoth were was used the used to PBS screened replenish as and using waterods replacement the independent lost water as PhyloChips. due aliquots previously to were describedof processed a in using nucleic the the acid same samplesample meth- concentration extraction. processing step After procedures processing, using the an withmatches. PBS Amicon the The controls Microcon showed replacement addition no YM-100 waterthe sign however, filter array. of displayed Because prior a positive the OTU replacement variety to water of was positive prepackaged and matches sealed on in unique blad- be representative of the originalronmental sample. samples This since is the an quantity unfortunatenot of necessity produce unamplified in robust DNA most results. is envi- relatively small and would loChips and to characterize background levels of 16S rRNA present in the collection Brodie et al., 2007. to retrieve the 16S-rRNAsidered considered present to if be at present leastset in 92% were the of positive. its sample. assignedmean A An probe-pairs noise probe-pair for OTU is threshold its was corresponding considered (MT). con- the probe positive noise The if value MT generated its is forDue signal each set to value PhyloChip according potential during is competitive to: processing above amplification (Brodie the MT processes et al., this 2007). relative abundance may not an Ultraclean Microbial DNAPolymerase Isolation chain reaction kit (PCR) (MOtractions. amplification Bio was Laboratories The then Inc., PCRcon performed Carlsbad, YM-100 products on CA). filters were the and cleanedfor DNA then preparing ex- and quantified the concentrated on collected a using samples UV for Amicon analysis spectrophotometer. Micro- on The the procedures PhyloChip were adapted from organisms. biochemical tests used in this study to help characterizetage isolates. of the 16Sfound rRNA in gene an that environmental all sample.the bacteria There chip. possess are to Operational greater identify taxonomical thanwithin bacterial units 500 the 000 components (OTU) 842 probes represent subfamilies arrayed groupings identified on to of by at similar DeSantis least et species 11 al.,dissimilar probes 2003. to (24 Each sequences probes OTU outside on corresponds ria average) the can thought OTU. be to In phylogenetically be this classified prevalent way, based in unknown on that environmental OTU their bacte- but similarity to known categories of dia types available, and if thehave full yielded spectrum additional of isolates; thesesary could however, to be due limit employed, this to both study the thebut may protocols broad study implemented group limitations, and in the it orderthe media was to study. selected neces- satisfy for This the use same to overall approach goals a was small without applied exceeding to the capacity the of selection of basic and specialized salt conditions, respectively.and The leucine amino ability peptidase of (LAP)production was the of determined bacteria gas by to additionalwere was separate produce then determined tests. catalase, compiled using The along indole presence Durham with or tubes. the absence gram of Thewere stain set growth results aside reaction, on of to colony several be these morphology, stored di tests in and glycerol the at time and logistics. There are a wide range of incubation conditions and specialized me- characterized using additional biochemical tests andAgar, MAC, selective motility media. test Simmons medium, Citrate Triple Sugarto Iron (TSI) examine and 6.5% the NaCl ability Agarnegative were of used organisms the and isolatesassess characterize to the utilize the citrate ability ability and to to ammonia, ferment ferment screen carbohydrates, lactose, for and gram assess assess motility, the ability to grow in high 5 5 25 15 20 10 25 15 20 10 Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | ecting erence ff ff cial National ffi er, 1995). Observations by ff 6732 6731 ects of meteorology on bacterial aerosol have been noted in ff 76.897 was used as the collection point on the JHU/APL cam- − erences may have been due to changing weather conditions a ff Initial isolate counts from the cultured samples yielded 41 unique isolates. Of the Although the diversity, measured by the microarray analysis, appears to increase The National Oceanic and Atmospheric Association (NOAA) Hybrid Single-Particle 41 isolates, 9 exhibited growthphology on consistent SDA, along with with fungal macroscopicnot and further isolates, microscopic characterized. and mor- Additionally, werewere several therefore initially isolates classified found thought within asstain to each fungal and be sample and that biochemical separate testsre-categorized organisms, results as were the after found same further tothe organism. isolation. re-categorization display of similar After These like the gram unique isolates isolates characteristics exclusion within were were of found samples, then the in a this fungal total study isolates (Fig. of and 3, 28 Tables 2 viable and isolates 3). with but Burkholderiales is foundwithin consistently this to phylum. represent Firmicutes greaterrepresenting than is anywhere the 11% from second of 8.6 most 16SNot dominant rRNA to surprisingly, phylum Bacilliales, 24.2% in Lactobacilliales of these and70% the samples of Chlostridiales mitochondrial the represent 16S RNA greater rRNA than in signatures identified the in samples. this phylum. in diversity between samplesindicate that collected some 4 organisms, or andbacteria, the 5 aerosol are particles November more associated suitable (Fig. with as 2 specific CCN types than and of others. Table 1)over may the course ofbulk the of 16S four rRNA sample in theto periods, samples. represent there Throughout 47.6 all are to the 63.8% awithin samples of Proteobacteria few this all are phylum phyla bacterial found that that 16S dominate rRNA represent the found sample the in vary the considerably samples. from sample The to orders sample, cate light precipitation periodicallytinuing beginning between at 10:18 least andairborne until 10:54 microorganisms EST 22:00 through and EST. con- collection Thistheir of incorporation precipitation these into organisms the may cloud by havenuclei and falling subsequently (CCN) resulted droplets, precipitation or by as in cloud simply the condensation by loss suppressing of the source of these particles. The di Weather Service observations from Baltimore/Washington International Airport indi- tional Weather Service NEXRAD datathe (not shown) state indicated of scattered Maryland showers across throughout the day on 4 November 2008, and o lection periods. Onewith can each see consecutive the collectionthough increase but collections not also one only withand in and the time, the number two the number di of are of phylathe similar positive environment. represented. to OTUs The collections Al- e previous three studies and (Brodie four etstudy in al., personnel 2007; location indicate Lighthart thatthe and precipitation first Sha was aerosol observed sample at collection the (05:30 collection to site during 09:30 EST on 4 November 2008). Na- 3 Results Phylogenetic data from allTable 1) four indicate aerosol that collection the periods biological on diversity is the increasing 4th over and the course 5th of (Fig. the 2, 4 col- Rolph, 2003) wassimilation used System to (EDAS) 40 generate kmof meteorological 36 dataset, air h to collected indicate back-trajectories, where duringlongitude using the these of parcels the 39.1665, experiments Eta travelledpus. prior Data The to runs As- were collection. initiatedand at A at 03:00 latitude UTC 11:00 for and UTC theinitiate collections for the between the model 05:30 was and collections 09:30 were 10 between m, collected. 13:30 to represent and the 17:30. height of The the height mezzanine used where to samples the refill water were subtracted outples. as a A background phylogenetic signal tree fromusing all of the the the Interactive processed Tree identified sam- of bacterial Life (Letunic orders and was Bork, created 2007). andLagrangian displayed Integrated Trajectory (HYSPLIT) model (e.g. Draxler and Rolph, 2003; signal were not easily implemented. Because of this, all positive OTU matches found in 5 5 25 15 20 10 25 15 20 10 Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | er, 1995; Mancinella ff erent days. It should be noted, that due to ff 6734 6733 erent time periods. An example of this can be seen ff erentiation of basic isolate characteristics (Fig. 3b, Table 3). Of the bacterial iso- The biochemical tests and selective media utilized in this study provided adequate Despite the limitations, it is clear that there is a highly diverse culturable microbiolog- Many of the isolates cultured initially appeared to be of low culturable concentrations ff cultivate these bacteria, the resultsisms. are The virtually mean identical across to the that four of samples the indicates cultured that organ- 46% of the bacterial load was and Shulls, 1978)studies find (e.g. a Brodie et preponderancebacteria al., of dominate 2007) the gram-positive tend nucleic bacteria, to acid.negative, 54% indicate while gram-positive The and that present 4% molecular the gram study variable.68% more indicates The to that PhyloChip fragile 85% results 42% of gram-negative suggest the were that are bacterial gram- phyla gram-positive found are (assuming typically gram-negative, onlyIf while the Firmicutes 15% to contribution and 32% frombers Actinobacteria are Proteobacteria are generally is considered gram-positive). removed to be from functional the anaerobes and analysis, no since attempt was its made mem- to 4 Summary and conclusions Qualitatively, the comparisons between thethe properties bacteria of identified the bythat culturable ribosomal have bacterial examined RNA culturable aerosol bioaerosols and (e.g. agree Lighthart with and previous Sha studies. Studies tured from separate samples collected onseveral di of the limitations discussedpresent below, but additional not common cultured isolates in mayof this have two study. been 4 Additionally, h this sample studysample collections only taken collections consisted back of taken to two over back. sets common a It isolates, broader is and possible time that potentially periodas a reveal would environmental higher trends conditions yield number associated or of a with times greater of their amount collection. prevalence of such chemical characteristics weretween also samples useful collected during for di when identifying looking common at isolates, theisolates even results displayed recorded the be- same for growth isolates characteristics,biochemical colony 2–4 morphology, reactions gram and stain, indicating 3–6 and that (Tables 2 they and may 3). be isolates Both of the same organism cul- fermenters, 78.2% were non-lactose fermenters. These basic morphological and bio- 30.4% grew under 6.5% NaCl conditions, 5.9% produced gas, 21.7% were lactose bacteria that were gram negative rodsrods varied between between 28.6% 20% and and 66.7%, 42.9%the gram and positive four gram indeterminate positive organisms coccito were between 17:30 found ET 0 during on and the 4 40%. secondwere November (Table isolated collection 2). Three during from of The that largest 13:30 collection number period. of distinct colonies, thirteen, di lates subjected to thesetive, tests, 43.5% 73.9% were were LAP catalase positive, positive, 17.4% 8.7% grew were on indole citrate posi- agar, 65.2% displayed motility, 33% gram positive rods, 21%consistently gram variable gram positive stains cocci (Fig. 3a, andconsidered Table 2). 4%, indeterminate The despite were 10.8% unable controls, of to displayed the befurther colonies removed that testing. from were the media, Over which the30% precluded and 4 the collections, bacterial the percent fungal varied percent between varied 53.8% between and 14.3% 85.7%. and The percent of the collection by the OMNI 3000.present Because in of the this, environment itviable that is by possible would the that collection have there system been were and culturable, organisms therefore but not were isolated inical rendered this load non- study. present in thethe characterized isolates samples. recovered were Over foundbacterial all to and four be 10.8% samples conclusively were taken, fungal, indeterminatethe 24.3% isolates 64.9% but of that were likely were conclusively bacterial known to in be nature. bacterial, Of 42% were the found 64.9% to be of gram-negative rods, as only 1 CFU of eachtypes. isolate appeared after Additionally, the there initialon incubation were a on several the single various instances media media growth in type on which as all non-selective 1 an CFU media.culturable isolate after These organisms indications only the may of be initial appeared potential the culture, low result concentrations of but of more after fragile replating organisms not displayed surviving aerosol 5 5 25 15 20 10 25 15 20 10 Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | http://www.arl.noaa.gov/ready/hysplit4. ective probes (CASCADE-P) using 16S ff 6736 6735 cult to rectify by comparing disparate studies. ffi We would like to acknowledge Bernard Collins and Michael Wagner of the Gene Array Core Facility at the Malaria Research Institute of The ff orts and support, Dorothy Paterno for critical review of the early findings, and ff ), NOAA Air Resources Laboratory, Silver Spring, MD, 2003. tory) Model access via NOAAhtml ARL READY Website ( Short-term temporal variability inMicrobiol., 74, airborne 200–207, bacterial 2008. and fungal populations, Appl. Environ. rDNA, Bioinformatics, 19, 1461–1468, 2003. cation and taxonomic classification ofotic environmental origins DNA from using both a prokaryotic microarray, FEMS and Microbiol eukary- Lett., 245, 271–278, 2005. regions, Appl. Environ. Microbiol., 39, 6–12, 1980. sequence construction for automated design of e G. L.: Urban aerosols104, harbor 299–304, diverse 2007. and dynamic bacterial populations, P. Natl. Acad. Sci., Initially, the broad diversity of the organisms found in the aerosol samples was alarm- Proteobacteria and Firmicutes represent the dominant two phyla throughout all the Hua, N.-P., Kobayashi, F., Iwasaka, Y., Shi, G.-Y., and Naganuma, T.: Detailed identification of Draxler, R. R. and Rolph, G. D.: HYSPLIT (HYbrid Single-Particle Lagrangian Integrated Trajec- Fierer, N., Liu, Z., Rodriguez-Hernandez, M., Knight, R., Henn, M., and Hernandez, M. T.: DeSantis, T. Z., Stone, C. E., Murray, S. R., Moberg, J. P., and Andersen, G. L.: Rapid quantifi- DeSantis, T. Z., Dubosarskiy, I., Murray, S. R., and Andersen, G. L.: Comprehensive aligned Cronholm, L. S.: Potential health hazards from microbial aerosols in densely populated urban Acknowledgements. for their e Daniel Portwood for his review ofand the written the work. other We would sta Johns also Hopkins like University to Bloomberg thank School Anne E. of Jedlicka Public Health. References Brodie, E. L., DeSantis, T. Z., Moberg Parker, J. P., Zubietta, I. X., Piceno, Y. M., and Andersen, to specific locations.treme Further environments such study as of factorydetailed urban smoke stacks genomic bacterial and profiling communities, sewage may treatment especially provideand plants, in useful using other source forensic ex- attribution information applications. for pollution control provide additional evidence to support attribution of pollutants or hazardous emissions ties, this information may be used to help identify the source location of pollutants and of the phyla thatvides should a have potential had explanation of oceanicOne the exciting origins. source implication of The of therecord these back-trajectory ribosomal of findings analysis signatures their is of pro- history. that theselarge phyla. It all desert is air sandstorms well samples may known will carrythat from carry bacteria hazardous past to a bacteria studies distant biological that locations may1980). extreme (Hua be events et transported This such al., study locally as 2007)dence and indicates over of 100 that the s microbial even of communities underSince within meters normal the the (Cronholm, urban path weather environment of conditions, is the genetic sampled likely air to evi- mass have may a exist. number of unique microbial communi- collected (Fig. 4). Thisevidence is of consistent both with soil 16S and rRNA oceanic found origins. in the samples,ing. which show After comparison withdence the was culturable gained fraction in of the the results; microorganisms, however, some this confi- did not explain the presence of many that may be important toboth consider. common soil For bacteria instance, and Acidobacteriathat are and both at Actinobacteria found are in least all some fourfrom air soil. fraction samples. Although of This a may the small indicate mycetes fraction aerosol and of collected Cyanobacteria each in sample, indicates thean that these presence open some samples of water of the was source, phyla the generated possiblyparcels Plancto- aerosol oceanic. had in The an these back-trajectory samples oceanicboth analysis had origin the indicate Atlantic east that ocean to all and north-east the land of mass New at Jersey heights close and to traveled the across ground before being dently, the results of theprevious studies molecular of analysis the and samecontrast culture type. that analysis Taken has together generally been these agree di results with present an interesting samples (Fig. 2, Table 1); however, several other phyla are represented in the samples gram-negative and 54% gram-positive (based on the assumptions above). 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I., and Sneddon, J.: Recent applications of laser-induced break- Kesavan, J. and Schepers, D.: Characteristics and Sampling E 5 5 30 25 20 15 10 Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | ActinomycetalesBifidobacterialesCoriobacteriales 0.00Rubrobacterales 0.00Unclassified 0.00 50.00 82.61 0.00 0.00 8.70 0.00 84.85 1.52 1.52 68.25 4.55 4.35 9.52 1.59 3.17 0.00 7.94 Chloroflexi-1bChloroflexi-1fUnclassified 0.00 0.00 100.00 0.00 0.00 0.00 8.33 0.00Chroococcales 25.00Nostocales 5.88 Plectonema 0.00 5.88 23.53 Prochlorales 0.00 0.00 100.00 0.00 0.00 0.00Lactobacillales 0.00 0.00Unclassified 33.30 0.00 27.78 7.69 0.00 7.69 Anaeroplasmatales 12.90 7.69 2.78Mycoplasmatales 5.56 7.69 0.00 20.93 3.23 6.45 19.08 0.00 0.78 0.00 0.00 1.97 1.32 0.66 AzospirillalesBradyrhizobialesCaulobacteralesConsistialesEllin314/wr0007Ellin329/Riz1046 0.00 0.00Rhizobiales 4.94RhodobacteralesRickettsiales 4.41 0.00 1.76Sphingomonadales 0.00 0.00 1.76Unclassified 1.23 3.94 0.88 1.52Ellin6095/SC-I-39 1.32 0.00 1.32 8.64 1.21MethylophilalesMND1 2.47 clone group 3.52 3.72 0.61 1.06 Neisseriales 0.91 6.17 0.61 7.49 1.60 Nitrosomonadales 0.00 1.23Rhodocyclales 3.08 2.42 0.53 Unclassified 1.23 0.00 1.60 7.27 0.53 5.76 0.44 6.17Bdellovibrionales 0.00 1.52 3.72 dechlorinating 0.44 0.00 clone 0.00 group 10.90 3.99 Desulfobacterales 0.30 4.55 1.23 0.00Desulfovibrionales 1.76 2.39 Desulfuromonadales 0.30 0.00 0.00 0.61 0.00EB1021 group 0.27 3.72 1.76 0.44Myxococcales 0.00 1.21 1.23Syntrophobacterales 0.27 0.00 0.00 0.44 1.06 0.00Unclassified 1.82 0.30 5.73 1.86 0.00 0.00 0.88 0.27 0.30 0.00 0.00Aeromonadales 1.86 0.27 1.23Alteromonadales 4.55aquatic 3.03 2.20 0.61 clone 0.27 group 0.27 0.00Chromatiales 1.23 1.32Ellin307/WD2124 3.99 1.23Enterobacteriales 3.19 1.52 0.80 3.70 0.00 0.00Legionellales 1.76 1.21Methylococcales 0.00Oceanospirillales 1.60 0.00 2.64 0.53 1.23 0.00Pseudomonadales 34.57 1.52 2.66 Shewanella 0.30SUP05 0.00 4.85 0.00 3.96 1.32 3.70Symbionts 0.00 1.06 0.00 4.94ThiotrichalesUnclassified 1.06 0.00 1.86 0.44 10.61 0.91 3.08uranium 0.80 waste clones 1.32 0.44Xanthomonadales 0.00 0.27 2.39 0.61 1.60 2.12 0.00 1.52 0.00 2.12 0.00 1.23 0.00 0.00 1.23 1.06 1.86 0.00 0.53 0.44 1.33 0.88 0.00 0.30 2.64 1.76 0.00 0.61 1.21 0.61 0.00 0.61 1.21 0.27 0.53 1.86 0.53 1.86 1.60 6740 6739 Acidobacteria-10Acidobacteria-4 UnclassifiedAcidobacteria-6Acidobacteria-7 Ellin6075/11-25Solibacteres UnclassifiedUnclassified Unclassified 0.00 0.00 Unclassified 0.00 5.00 Unclassified 5.00 0.00 10.00 0.00 0.00 0.00 0.00 0.00BD2-10 group 5.56 15.00 2.33 9.30 0.00 10.00 Unclassified 20.93 5.56Flavobacteria 11.11Sphingobacteria 2.33 Unclassified 50.00 6.98 Sphingobacteriales Flavobacteriales 4.65 66.67 4.35 11.11 Unclassified 85.71Unclassified 0.00 1.52 66.67 11.11 11.11 1.59 Unclassified 67.74 14.29Chloroflexi-3 6.45 Chloroflexi-4 11.11 50.00DehalococcoidetesThermomicrobia Unclassified Roseiflexales 9.68 Unclassified 0.00 Unclassified Unclassified 0.00 0.00 50.00 Unclassified 0.00 0.00 0.00 27.27 40.00 9.09 0.00Unclassified 0.00 16.67 0.00 8.33 27.27 0.00 17.65 Unclassified 5.88 16.67Clostridia 11.76 5.88 0.00Desulfotomaculum 17.65 Mollicutes Unclassified Clostridiales 0.00Symbiobacteria 0.00Unclassified Acholeplasmatales 33.30 13.89 Symbiobacterales 0.00 9.68 2.78 23.08 Unclassified 22.58 0.00 2.33 6.45 31.78 0.00 1.55 38.16 2.63 1.55 6.45 0.66 1.32 2.33 1.32 Unclassified Unclassified 100.00 0.00 0.00Betaproteobacteria 66.67 Burkholderiales 11.11Deltaproteobacteria 15.42 AMD clone group 14.55 0.00 16.76 0.00EpsilonproteobacteriaGammaproteobacteria Campylobacterales Acidithiobacillales 1.52 12.35 1.33 0.00 7.93 0.00 6.36 0.91 6.12 0.27 Nov 4 AM Nov 4 PM Nov 5 AM Nov 5 PM Nov 4 AM Nov 4 PM Nov 5 AM Nov 5 PM Nov 4 AM Nov 4 PM Nov 5 AM Nov 5 PM Nov 4 AM Nov 4 PM Nov 5 AM Nov 5 PM Continued. Taxonomic distribution of OTUs identified in the four collected aerosol samples. Actinobacteria 1.34AD3 6.41AquificaeBacteroidetes 10.08 8.06BRC1 6.04Caldithrix 0.67Chlamydiae Actinobacteria 1.95Chlorobi 0.28 AcidimicrobialesChloroflexi 4.12 0.00 0.00 0.00 0.67 0.15 3.96 1.34 0.00 0.00 0.00 0.56 0.13 2.68 Bacteroidetes 0.28 0.15 0.15 6.06 0.31 Unclassified 0.13 3.06Coprothermobacteria Bacteroidales 0.31 0.13 0.13 7.94 0.13Cyanobacteria 0.00 Unclassified Aquificae 1.83 11.11 0.64 Unclassified Chlamydiae Unclassified 0.00 0.67 2.17 100.00 0.00 Chlorobia AquificalesDeinococcus-Thermus 0.15 Caldithrales 100.00 Chlamydiales Unclassified 0.56DSS1 0.00 Anaerolineae 11.11 100.00Firmicutes 0.13 0.00 0.00 0.00 Chlorobiales 100.00 0.46 0.56 16.13 Chloroflexi-1a 100.00 100.00 Unclassified 0.00 0.00 0.00 1.66 0.31 0.00 50.00 24.16 0.00 100.00 100.00 Unclassified 0.00 100.00 100.00 Cyanobacteria 0.38 36.36 100.00 8.64 0.00 100.00 100.00 50.00Gemmatimonadetes Chloroplasts 25.00 100.00 Unclassified 0.00Lentisphaerae 19.69 0.00 0.00Natronoanaerobium 60.00 5.88 23.53 0.00 0.00 0.67 Unclassified 0.13 0.56 0.00 Bacilli 100.00 100.00 0.56 Unclassified 0.00 0.61 0.28 33.30 100.00 0.61 100.00 0.77 0.15 Unclassified 46.15 Bacillales 100.00 0.64 Unclassified 0.13 0.00 100.00 Unclassified 47.22 Unclassified Unclassified 0.00 32.26 Unclassified Unclassified 0.00 0.00 38.76 100.00 100.00 100.00 0.00 32.89 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 PhylumAcidobacteria 0.67 5.57 Phylum % of OTU 2.75 5.50 Class Acidobacteria Acidobacteriales 100.00 Order 55.00 77.78 53.49 Order % of Phylum PhylumNC10NitrospiraOP10 0.00OP3 0.00OP8OP9/JS1 Phylum % of 0.67 OTU 0.28Planctomycetes 0.28 1.34Proteobacteria 0.00 0.00 0.28 0.31 53.69 0.00 0.15 0.84 0.28 63.79 0.56 0.46 0.51 Class 0.28 0.26 0.31 50.08 0.15 0.31 0.38 Nitrospira 0.15 NC10-1 0.64 47.57 0.13 0.26 CH21 cluster 0.00 Planctomycetacia Alphaproteobacteria Order Unclassified OP9 Nitrospirales Acetobacterales Unclassified Unclassified Planctomycetales Unclassified Unclassified 0.00 100.00 Unclassified 0.00 0.00 100.00 Unclassified 1.32 0.00 100.00 100.00 100.00 0.00 0.61 100.00 100.00 0.00 100.00 100.00 Order % of 100.00 100.00 Phylum 100.00 0.00 1.06 100.00 100.00 33.33 100.00 100.00 100.00 100.00 0.00 100.00 100.00 Table 1. Table 1. Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | 6742 6741 Unclassified UnclassifiedUnclassifiedVerrucomicrobiae 0.00 Unclassified Verrucomicrobiales 1.32 0.91 75.00 0.00 71.43 1.06 0.00 80.00 100.00 60.00 66.67 -white circular entire glistening-white raised circular Chocolate entire dry 24 GN rods raised Chocolate 24 N/R ff ff Nov 4 AM Nov 4 PM Nov 5 AM Nov 5 PM Nov 4 AM Nov 4 PM Nov 5 AM Nov 5 PM Size Color Shape Edge Surface Elevation Media Time (hr) Stain Shape Morphological and gram-stain properties of cultivated isolates from the four collected Continued. PhylumSPAMSpirochaetesSynergistesThermodesulfobacteriaTM6 0.00TM7 0.00 0.00 0.00 0.67Unclassified Phylum % of 0.00 OTUVerrucomicrobia 0.00 0.28 0.28WS3 0.46 0.00WS5 2.68 1.34 0.13 0.31 0.61 0.00 1.66 0.00 Class 1.95 1.95 Thermodesulfobacteria 0.26 0.77 0.28 Thermodesulfobacteriales Spirochaetes 0.00 0.00 0.76 1.22 0.00 Unclassified Unclassified 0.15 0.00 0.13 0.00 0.64 1.41 0.56 0.38 Spirochaetales 0.00 Order Unclassified 0.00 Unclassified Unclassified Unclassified Unclassified 0.31 TM7-3 0.00 100.00 0.00 0.13 0.13 Unclassified Unclassified Unclassified 0.00 100.00 0.00 Unclassified Unclassified 100.00 Unclassified 100.00 100.00 100.00 0.00 100.00 25.00 100.00 100.00 Unclassified Order 100.00 % Unclassified of Phylum 100.00 100.00 0.00 28.57 0.00 100.00 20.00 0.00 100.00 0.00 100.00 0.00 40.00 0.00 100.00 100.00 0.00 33.33 100.00 0.00 100.00 100.00 Isolate Number1–1, 21–31–41–51-6 small1–71–8 large1–9 large white1–10, 11, medium 131–12 opaque medium o 2–1 orange medium pinpoint2–2 yellow pinpoint gray/white2–3 clear small2–4 clear small filamentous2–5 Colony Morphology circular filamentous white2–6 small rough2–7 circular medium white irregular2–8 large circular filamentous orange white2–9 medium rough entire entire2–10, circular 13 medium circular gray/white2–11 entire opaque small yellow/gray2–12 large entire filamentous rough2–14 smooth medium entire filamentous small white3–1 circular glistening large rough gray circular3–2 gray filamentous circular smooth medium3–3 raised white irregular smooth filamentous entire small irregular3–4 o white entire smooth large3–5 TSA, entire SDA entire raised3–6 entire medium opaque smooth3–7 medium orange circular smooth TSA, white4–1 SDA small glistening glistening raised circular white4–2 raised circular raised medium smooth4–3 filamentous entire large raised SBA white white4–4 filamentous SBA filamentous TSA, large raised SDA entire entire4–5 rough filamentous raised large circular 24 SBA4–6 yellow Growth rough very raised Conditions smooth large TSA circular4–7 white large SBA white filamentous glistening rough entire yellow, darker medium 48 TSA center raised filamentous filamentous entire small raised raised circular rough filamentous raised bright yellow orange pinpoint rough circular circular SBA convex glistening small TSA, SBA Chocolate 48 SBA white opaque entire small circular rough N/R edges, TSA smooth Gram center entire entire Stain 24 24 circular circular opaque convex circular raised entire yellow raised N/R glistening raised 24 raised Chocolate, 48 SDA entire circular Chocolate glistening smooth entire 24 entire GP 24 GN raised SBA TSA Chocolate, circular 24 SDA glistening N/R rods circular rods/cocci raised raised entire Chocolate, SDA GP smooth smooth circular raised rough 48 edges, GN unable smooth entire GP to center pick 24 TSA TSA rods 24 48 entire circular raised rods TSA, rods rough 24 Chocolate, edges, entire SBA, smooth SDA unable center to raised 48 pick GN smooth 24 TSA, 24 raised entire SDA raised GP smooth 24 GP raised SBA, small SDA rods glistening GP TSA raised cocci clusters rods TSA 24 48 glistening TSA, GN rods Chocolate N/R Chocolate raised raised rods N/R GP SBA N/R TSA 24 48 24 N/R cocci unable clusters to raised pick 24 24 raised raised GP GN TSA raised TSA 48 24 Chocolate rods rods 24 GP Chocolate cocci N/R clusters GP GN GN N/R 24 48 small rods rods rods 48 GP GP 48 48 48 cocci rods clusters GP GN GN Variable cocci clusters rods rods aerosol samples. Table 2. Table 1. Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | NG –– NG NG –– NLF NG – NG – NG + + + + + + + + NC – – LF A/A N/R N/R NG K/A A/A -K/K - - N/R NLF NG A/A – –A/A K/A NG K/A K/A N/G – – LF NC gas N/R N/R NG NC gas N/R N/R NG NC gas – – LF K/K gas N/R N/R NG – K/A – – NLF + + + + + + + + + + + + –– – NC gas – – NG – – – – – – – – NC – – LF + + + + + + 6744 6743 + + + + + + + + + – – – –––– – ––– – – – A/A – – – ––––– K/K – – LF – ––– – – NC – – NG –––––– – – – A/A + + + ++ ++ + + + + + + + + + + + + + + + Biochemical properties of cultivated isolates from the four collected aerosol samples. Flow chart for processing collected aerosol samples. 1–10, 11, 132–10, 13 – – 4–5 4–7 3–4 4–2 4–4 3–13–33–5 4-3 – – – – – – – – – NC gas – – NG 3–6 Isolate Number Catalase1–5 Indole1–6 LAP Citrate Motility TSI NaCl Gas2–6 MacConkey 2–12 1–71–8 – – – – 1–122–5 – – 2–2 2–3 2–4 Fig. 1. Table 3. Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | lactose - Fermenters Non No Growth Lactose Fermenters gas K/A NC A/A N/G K/K No Results Negative 1 0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 6746 6745 (B) Mold, 27.03% Unknown, 10.81% Summary of the tested biochemical properties of all bacterial isolates 21.62% Gram Positive Rods, Rods, Positive Gram (B) Only, 5.41% Only, Gram Negative Negative Gram Morphological distribution of cultivated microbial isolates from the four collected 13.51% 18.92% Phylogenetic distribution of bacterial orders with OTUs that represent greater than 1% Cocci, 2.70% Gram Negative Negative Gram Gram Positive Cocci, Positive Gram Gram Negative Rods, Negative Gram (A) Fig. 3. (A) aerosol samples. from the four collected aerosol samples. of the OTUs found inthe the relative four abundance collected aerosol of samples. distinctTable 1. The OTUs outer from stacked each bar order chart in indicates each sample that is also shown in Fig. 2. Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | 6747 Thirty-six hour back trajectories from each of the four collected aerosol samples. The altitude is given adjacentsample indicates to that each all samples pointMapping travelled and denoted at satellite relatively on imagery low the generated altitudes using over trajectories. Google both Earth. ocean The and land. trajectory for each Fig. 4.