Detection of gram-negativeErwinia herbicola outdoor aerosols with pyrolysis-gas...

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FIELD ANALYTICAL CHEMISTRY AND TECHNOLOGY 4(2–3):111–126, 2000

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� 2000 John Wiley & Sons, Inc.

Detection of Gram-Negative Erwinia herbicolaOutdoor Aerosols with Pyrolysis–GasChromatography/Ion-Mobility Spectrometry

A. Peter Snyder, 1 Waleed M. Maswadeh, 2 Ashish Tripathi, 2 and Jacek P. Dworzanski 3

1U.S. Army Edgewood Chemical Biological Center, Aberdeen Proving Ground, Maryland 210102Geo-Centers, Inc., Gunpowder Branch, P.O. Box 68, Aberdeen Proving Ground, Maryland 210103Center for Micro Analysis and Reaction Chemistry, University of Utah, Salt Lake City, Utah 84112

Received 29 May 1999; revised 28 August 1999; accepted 14 September 1999

Abstract: Aerosol particulate species of the gram-nega-tive bacterium Erwinia herbicola (EH) were detected bystand-alone, analytical instrumentation in an outdoorwestern United States desert test site. The device con-sisted of an aerosol collector interfaced to a quartz-tubepyrolysis–gas chromatography/ion-mobility spectrome-ter (Py-GC/IMS). The detector is about the size of a shoe-box, that is, 12 � 9 � 6 in. Bacterial aerosols and back-ground particulates in the 2 to 10 �m-diameter rangewere collected by a 1000-l/min aerosol concentrator anddeposited onto a filter in a quartz tube. Rapid heating to350 �C in 5 s effected vaporization, and a portion of thepyrolyzate was directed into a GC column. The eluatewas detected by the atmospheric-pressure–based IMS.A distinct peak in the GC/IMS data window was used tosignal the presence of the EH bacterial aerosol. The sen-sitivity of this method was relatively good in that valuesdown to five EH-containing aerosol particles per liter ofair could be detected in approximately 2.5 min. � 2000John Wiley & Sons, Inc. Field Analyt Chem Technol 4:111–126, 2000Keywords: pyrolysis; ion-mobility spectrometry; fieldbiodetection; biodetection; aerosol concentrator; gaschromatography; biological aerosols; Erwinia herbicola ;bacteria

Introduction

Analytical investigations of aerosols have relied on a di-verse set of approaches over the last three decades from ex-perimental determinations of generated aerosols from bulksolutions to real time analyses of ambient outdoor particles.These investigations have included inorganic (salts) and or-

Correspondence to:A. P. Snyder

ganic particulates as well as bioaerosols that include micro-organisms, fungi, and pollen. An accounting of the mostprevalent techniques and instrumental methods appears con-structive with respect to the present analytical-detectionmethod of biological aerosols.

Traditional methods for the characterization of aerosolsconsist of sampling ambient air and collecting/concentratingthem on various matrices.1–3 These biological aerosol par-ticulates are then subjected to sample detection techniquessuch as polymerase chain reaction (PCR),1,2,4,5colony platecount or most probable number (MPN),1,4,6,7 biolumines-cence from inherent adenosine triphosphate (ATP),4,8phase-contrast microscopy,4 and immunoassay.1,4,9 The bacterialaerosol samples were characterized by these traditional de-tection techniques in either an off-line or on-line fashion.

Pseudomonas fluorescensbacteria were aerosolized anddirected to an agar plate with a laser sizing system placedafter the aerosol generator. This was an important develop-ment in that a relation could be produced between the totalnumber of particles and the number of bacterial-colony-forming units on the agar growth plate.10

Mass spectrometric methods have had a long and richhistory as analytical vehicles for investigating compositionalproperties of artificial and outdoor man-made aerosols aswell as ambient organic and inorganic aerosols under off-line or on-line analysis conditions.

Laser microprobe mass analysis (LAMMA) has beenused in an off-line fashion to investigate aerosol particles.Particles were collected or placed on a matrix or wire meshand were introduced into a vacuum. A microscope guides alaser beam to a selected particle or spot on thematrix surfacewhere ions desorb and are analyzed by a time-of-flight massspectrometer (TOFMS). Relatively low molecular weightspecies were usually observed, and known species mostly

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112 FIELD ANALYTICAL CHEMISTRY AND TECHNOLOGY—2000

represented the inorganic salt fraction of samples such asMycobacterium leprae,11,12 B. anthracis, B. thuringiensis,andB. cereus,13,14 as well as ionized species of particulatesincluding polyaromatic hydrocarbons (PAH)15 and salt spe-cies.16 Two recent review articles on LAMMA document theprincipal and extensive applications of TOF and Fourier-transform mass-spectrometer analyzers in the analysis of la-ser-generated ions of single aerosol particles17,18 that wereplaced or impacted on matrix supports.

The TOFMS field evolved to where an aerosolized sus-pension of biological particulates could be introduced into avacuum as single particles. This procedure, particle analysisby mass spectrometry (PAMS), used either a hot rheniumfilament to pyrolyze individual bacterial particles19–22 or alaser to desorb and ionize species from particles.22,23Thesebiological particles, includingPseudomonas putida,Bacilluscereus, andBacillus subtilisvar.nigerwere generated froman ethanol–water suspension. Linear quadrupole mass spec-tral determinations mainly produced unknown pyrolysisfragments and inorganic salt-derived species.

A similar system, developed by Gieray, Reilly, Yang,Whitten, and Ramsey24 used an ion-trap mass-spectrometerdetector. From a bulk water suspension, bacterial aerosolparticles were sensed and a trigger was provided by the par-ticles passing through two argon ion laser beams. An exci-mer laser ablated the particle in the ion trap so as to produceions. An improvement on this basic design was that of aTOF system25–27 replacing the quadrupole mass spectrome-ter designs. This allowed for faster mass spectral scanningof ions from particles generated from bulk suspensions ordirectly from laboratory ambient air. Salt particles as wellas tobacco smoke and soot were analyzed.

Hars et al. used a combined electrodynamic balance/ion-trap mass spectrometry technique for trapping and stabiliz-ing aerosolized particles of polystyrene andBacillus subtilisspores, followed by laser fragmentation/ionization to obtainmass spectra of the ions generated during a 450-mJ pulsefrom a Nd-YAG laser. They demonstrated the feasibility touse this technique for chemical and physical characterizationof single cells of microorganisms and other components ofrespirable aerosols.28,29In other work, an aerosol particle siz-ing laser was interfaced to a laser desorption-pyrolysis/ion-ization beam for TOFMS analysis on organic and inorganiccompounds.30,31

Prather et al. published a series of evolving articles withthe concept of size, aerodynamic diameter, chemical com-position and composition class, and temporal characteriza-tion of outdoor aerosol particles.32–39The centerpiece was atransportable aerosol concentrator, dual time-of-flight massspectrometer. Positive ions are analyzed in one tube and neg-ative ions, from the same particle, are analyzed in the secondtime-of-flight tube. As an example, over a period of fourdays, pyrotechnic explosives (fireworks) particlesweremon-itored in the atmosphere; monitoring sites were 0.5 and 3miles from the explosion sources.37 Further examples of thistechnology are the characterization of automobile emis-sions,36 where metals, oxides, hydroxides, and polyaromatic

hydrocarbons (PAH) were detected, and in the temporalmonitoring of the nitric acid to hydrochloric acid heteroge-neous chemistry that occurs in atmospheric aerosols over theocean– land-mass interface.39

Gas chromatography (GC)/MS has been used for the traceanalysis of bacteria and fungi in organic dust aerosols fromenvironments such as hospitals and homes40–44and biotech-nology processes.45,46 The air was continually sampled forbacteria for a period of 24 h, and the bacteria on the filterwere processed for the extraction of specific biochemicalcompounds for GC/MS analysis.

Biological aerosol analyses using analytical instrumen-tation were mainly relegated to controlled investigations inlaboratory settings as related earlier. Only a few investiga-tions can be found in the literature concerning the real-timedetection of bacterial aerosols in outdoor scenarios, and thedetection methods were primarily spectroscopic in nature.Real-time detection ofBacillusspore aerosols from dissem-ination of bulk suspensions in outdoor testing areas was ac-complished by a light detection and ranging (LIDAR) sys-tem. This is a remote detection system where a 1064-nmlaser beam was used to interrogate aBacillus subtilisvar.niger aerosol plume approximately perpendicular to thebeam.6,47 The backscattered radiation was collected by re-ceiver telescope optics and used in the determination of bac-terial aerosol presence.

The fluorescence of an aerosol ofB. subtilis from bulksuspension was detected as single particles with a fluores-cence particle-counter instrument in outdoor and under in-door, controlled conditions. An argon laser beam of 488 nminterrogated a beam of particles by monitoring emission inthe 530 to 550-nm range.48,49Flavin compounds in the bac-teria were surmised as the bacterial component responsiblefor the emission at 530 to 550 nm, and nonbiological inter-ferences displayed no fluorescence activity in the emissionbandwidth. Another report dealt with combining the 488-nmargon laser, which produces size-scattering and visible flu-orescence, with a 266-nm pulsed laser to produce 300–500-nm UV-VIS fluorescence of the proteinaceous bacterialcomponent50 from the generated aerosols. Similar experi-ments withB. subtiliswere shown with a 325-nm helium–cadmium laser excitation source with fluorescence monitor-ing at 420 to 580 nm.51

The first Py-GC/IMS analyses of biological materials, in-cluding Bacillus spores and nucleic acids, appear to havebeen reported by Meuzelaar, Kim, Arnold, Kalousek, andSnyder.52This was followed by systematic Py-GC/IMSstud-ies of various biopolymers relevant to bioagent detection53

as well as potential interferants, culminating in a recent PhD.thesis by Thornton.54

Prototype portable GC/IMS concept systems were shownto successfully separate headspace vapors from complex liq-uid mixtures of analytes.55–57

Laboratory testing of the prototype of the currentlyfielded Py-GC/IMS version was performed under controlledsample introduction of bacterial suspensions.58 Moreover, apreliminary presentation of a spore aerosol investigation in

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FIELD ANALYTICAL CHEMISTRY AND TECHNOLOGY—2000 113

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FIG. 1. Photograph of the complete XM-2 aerosol concentrator-Py-GC/IMS system.

the field with the prototype Py-GC/IMS was reported re-cently.59 Finally, a more comprehensive study of the detec-tion of outdoor aerosols ofBacillus subtilisvar.niger (BG)bacterial spores with Py-GC/IMS has recently been per-formed.60

The present investigation provides for the determinationof desirable properties of generated biological aerosols.Er-winia herbicola(EH) aerosols were investigated in an out-door desert scenario with an aerosol concentrator-pyrolysis-gas chromatography/ion-mobility spectrometer system(Py-GC/IMS). This system was used to experimentally de-termine the presence of bacteria in an aerosol plume (cloud).

Experimental Section

Figure 1 shows a photograph of the complete aerosol col-lector-Py-GC/IMS system that was used to collect and in-terrogate outdoor-released bacterial aerosols. Note that theaerosol collector, which is located in the upper half of Figure1, is relatively larger (25� 17� 13 in.) and much heavier(100 lb) than that of the detector system (Table 1). The smallinstrument on the lower right-hand corner is a Met-One par-ticle counter (Met One, Inc., Grants Pass, OR).

Figure 2 shows a display of the pyrolysis-gas chromatog-raphy/ion-mobility spectrometry (Py-GC/IMS) unit. Thisshoebox-sized Py-GC/IMS system is smaller in dimension,considerably lighter in weight, and has a lower power budgetthan that of the briefcase-sized system.60 The major com-ponents of the system are as follows: 1—12� 9 � 5-in.carrying case; 2—high-temperature three-wayGC-injectionvalve and pump connection to draw aerosol particulate ontothe quartz filter; 3—temperature-programmable GC ring;4—diaphragm vacuum pump; 5—electronic controlboards; 6—50-pin interface to PCMCIA data acquisitioncard; 7—Hitachi mini-notebook with data acquisition card(PCMCIA) from National Instruments (DAQ card-1200,Austin, TX 78730); 8—molecular sieve packs; 9—voltage-gated ion source and ion-mobility spectrometer componentsof airborne vapor monitor (AVM) (Graseby-Dynamics,Watford, Herts, UK); 10—coaxial cable for Ethernet/serialPC card communications; 11—dc power in; 12—aerosolinlet; 13—quartz filter aerosol collector in a quartz tube/pyrolysis source.

The pyrolysis module consists of a quartz frit that acceptsthe concentrated aerosol particles from a 1000 l/min aerosolcollector/concentrator. Typical aerosol concentration timeswere in the 1.5–2.0-min time frame. Pyrolysis (rapid heat-ing) to 350�C vaporized the solid particulate, and a portionof the pyrolyzate was admitted into a GC column. The eluteenters a63Ni ring ionization source of the IMS. A voltagegate pulses packets of ions into a drift tube of approximately4 cm at close to atmospheric pressure. The flight time of theions between the ion gate and Faraday plate detector char-acterizes the ions. Different molecular weights and cross-sections of ions primarily determine the drift time and degreeof separation of a mixture of ions.

Figure 3 presents details of the pyrolysis region and three-way injection valve: -in. OD� 0.03-in. ID glass-lined11—8

stainless steel tube; 2—high temperature, three-way injec-tion valve with in. Swagelock fittings (Baltimore Valve &1

8

Fitting, Baltimore, MD 21228); 3—glass-lined stainlesssteel, transfer line tube (ID� 0.02 in., OD� 0.0625 in.,Silcosteel from Restek Inc., Bellefonte, PA 16823);4—clean dry air; 5—vacuum for aerosol particulate collec-tion; 6—patented programmable GC ring oven; -in.17—8

Swagelock -in. tube reducer with -in. stainless-steel tube;1 18 16

8—high-temperature, stainless-steel GC column, 4 m�0.5 mm with 0.25�m methylsilicate coating (QuadrexCorp., New Haven, CT 06525); 9—injection liner withquartz tube, -in. OD� -in. ID � 72 mm long; 10—pyr-1 1

4 8


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TABLE 1. Experimental operating conditions of the Py-GC-IMSsystem.

Overall propertiesWeight (lb)Avg. power consumption (W)

Volume (ft3)Dimensions (in.)

10�120 (pyrolysis)60 (Normal run, no Py)0.3112� 9 � 5

PyrolyzerWireResistance (ohm)Tube diameter (mm)Length (mm)Pyrolysis time (s)Wire temp (�C)

Nichrom (0.015 in. OD)3.36 � 4654–10700–900 (estimated)

FilterTypeDiameter (mm)Particle size (�m)

Quartz micro fiber4�1.0

GC columnUltra-alloy stainless steel column (high temperature)Liquid PhaseLength (m)Inside diameter (mm)

Temperature (�C)Carrier gasFlow Rate (ml/min)Sample injection pulse (s)Injection valve temp (�C)

Methyl silicone (0.25 um)4.00.560–130 at 120�C/minClean dry air20 at GC column 60�C0.5–2.0 (select)150

Ion-Mobility Spectrometer:Ionization sourceGating pulse rate (Hz)Cell temperature (�C)Cell pressure (torr)Drift gasMode

63Ni, 10 mCi30 (internal)35520Clean dry arPositive ion

olyzer heater wire; 11—1/8-in.-diameter, 1�mquartz filter;12—retainer spring.

Aerosol particulates in the 2 to 10-�m diameter rangewere collected by a four-stage, 1000 l/min XM-2 aerosolconcentrator (SCP Dynamics, Minneapolis, MN 55432).The particulate is drawn out of the fourth stage at 250 to 300ml/min and onto the frit inside the quartz pyrolysis tube(Figure 3). The three-way valve (No. 2, Figure 2) is switchedto a vacuum pump (No. 4, Figure 1) in order to admit theparticulate onto the frit. The particulate was pyrolyzed at 350�C, and a portion of the pyrolyzate was injected onto the GCcolumn by the high-temperature three-way valve. A 1-spulse of pyrolysis products was admitted onto the GC col-umn, and clean, dry air was used as carrier gas. Molecularsieve packs (No. 8 in Figure 2) were used to scrub ambientrecirculation air. The eluate was ionized by the63Ni ring atthe entrance to the AVM ion-mobility spectrometer. Analyteionization is effected by proton transfer from reagent pro-tonated water molecule clusters. Table 1 provides analyticalparameter operation values for the Py-GC/IMS system.Temperature measurements of the pyrolyzer, three-way in-jection valve and GC were monitored by using type-K ther-mocouples connected to in-house constructed electroniccontrol boards.

The mini-notebook computer in the Py-GC/IMS systemwas remotely controlled from approximately 150 ft with theuse of PC ANYWHERE software (Symantec Corp., Cuper-tino, CA 95014) from a Samsung Sens Pro 500 laptop com-puter (Samsung, Korea) by way of an Ethernet coaxial cable(Black Box Corp., Lawrence, PA 15055).

The acquisition of GC/IMS data was accomplished byusing National Instrument software that was customized inhouse, and the data stream was converted into ASCII. TheASCII data were postprocessed with a Fortran programwrit-ten in house.

Figure 4 shows an enlarged photograph of the -in. quartz18

microfiber filter after multiple pyrolysis cycles. The whiteedge of the filter [1, inset to Figure 4(a)] is held by a retain-ing spring (2) on the quartz frit inside the aerosol collector/pyrolyzer tube (3). In Figure 4(b), the pyrolysis wire isshown wrapped and cemented (high-temperature Sauereisenceramic cement No. 8 powder from Scientific InstrumentalServices, Inc., Ringoes, NJ 08551) (4) around the quartzfilter/frit assembly and extends approximately 0.5 in. fromeither end of the quartz filter/frit.

Details of the three phases of a typical cycle are shownin Figure 5. The aerosol collection phase usually was be-tween 1 and 2 min. This was followed by a 15-s period fordrying of the aerosol sample deposited on the filter. Thepyrolysis event then occurred (9 s) (scan function No. 3) andsome time was necessary for switching flows (12 s) (scanfunction No. 4). The sample drying and GC analysis phasesin Figure 5 have similar airflows throughout the system. Atransitional airflow takes place between these two phases.This is approximately 12 s in duration and includes differentairflows to sweep the pyrolyzate out of the pyrolysis regionand to introduce sample to the system in the GC injectionpulse. The pyrolysis event consists of 4 s of heating and 5 sfor temperature equilibration inside the pyrolyzer tube. Thetemperature programming of the GC column begins (scanfunction No. 6) at the end of the pyrolysis event (scan No.3), and a portion of the pyrolyzate is injected into the GCcolumn (No. 5 scan function) for approximately a 1-s du-ration after initiation of the GC temperature programmingramp.

A vehicle-mounted Micronair agricultural sprayer assem-bly disseminated bioaerosols at approximately 800 m fromwhere the Py-GC/IMS biological detector was placed in thedesert. A slurry of dry BG spores in water, stored in a res-ervoir tank, was drawn through a tube and forced into aspinning wire mesh. The spinning mesh partitioned the liq-uid slurry into approximately 50-�m droplets, and thesedroplets were disseminated into the air by a fan. The bac-terial particles evaporated water as they traversed the 800-m distance and resulted in particle diameters in the 0.7 to10-�m micron range.

Bacterial aerosol particle counts were enumerated by thetest personnel, and this was accomplished by interfacing aslit sampler to Petri dishes situated on automated revolvingdisks.

A propylene gas tracer was released, and then the biolog-

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FIG. 2. Picture of the Py-GC/IMS shoebox-sized unit. Refer to the Experimental section for the legend.

ical aerosol was released at a known time from theMicronairassembly after the tracer gas release. If the propylene gaswas detected at the test grid, the slit sampler was turned onand admitted air samples onto an agar Petri dish. The timethat the propylene gas tracer was released and the time ofrelease of the bioaerosol was known to the test directors andunknown to the instrument operators. A small pie section ofthe Petri dish was exposed to the aerosol slit sample collec-tor, and a fresh section on the agar plate was exposed every4 s for a total time of 2 min per Petri dish. Eight separatePetri dishes, each with their own slit sampler, were used fora total of 16 min of aerosol collection during a trial.

The Petri dishes provided an aerosol record of the tem-poral presence of bacterial particles with a 4-second reso-lution with respect to the presence/absence of a biologicalcloud. The slit sampler provided no discrimination as to thesize of the particles that impacted on the Petri dishes. Thus,a wide range of particle sizes was directed onto the Petridishes, including the 0.7–10�m respirable-particle sizerange. The dishes containing trypticase soy agar were in-cubated at 37�C for 24 h, and colonies were enumerated forEH. Each bacteria-containing particle that impacted on theagar plate produced a spot or colony-forming unit (CFU)upon growth of the bacteria during incubation. The particle


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FIG. 3. Schematic of the aerosol introduction pathway, pyrolysis region, vapor transfer, and GC three-way injection valve region. Refer to the Experimentalsection for the legend.

can contain one or more individual bacterial cells, however,only one CFU will be produced. Thus, a graph was con-structed that described time versus CFU per liter of air oranalyte-containing particles per liter of air (ACPLA), andthe graph was subjected to a smoothing routine. The inter-pretation of an ACPLA is based on the volume of a sphericalparticle of a given diameter and the maximum number ofanalyte organisms that can fit in the spherical volume.61Usu-ally, nonbiological organic and inorganic salts and debris/growth media are also contained in a representative aerosolparticle generated from a bacterial suspension. This com-plexity in an aerosol particle does not allow for a straight-forward calculation of the number of bacteria in a givenparticle size.

Aerosol collectors such as a Met-One device measure to-tal particles per liter of air (PLA), whereas an agar dish onlymeasures viable bacteria-containing particles. Thus, theMet-One aerosol information is usually equal to or higher in par-ticle counts than that of the Petri dish method. The Met-Onewas programmed to measure particles of 1 to 10�m in di-ameter. The data output was smoothed by a three-pointsmoothing routine. This reduced the amount of data to one-third of that of the original data set. The reduced set wasthen subjected to a standard cubic spline smoothing routine.

During the four-week series of aerosol trials, the sameGC column was used. The pyrolysis quartz filters were re-

placed after every day of operation, because after 8 to 10 hof outdoor trials, the filters experienced a buildup of pyro-lyzate char and tar in the form of gray–black deposits. Nosignificant GC column degradation was noticed (i.e., no sig-nificant shift of analyte retention time), and the same air-scrubbing molecular sieve package was used.

Results and Discussion

The XM-2 aerosol concentrator-Py-GC/IMS system wasplaced on a table in an outdoor desert site in the westernUnited States. A trial window typically encompassed a 20min–1.0 h time frame, and a bacterial aerosol was releasedfor a 3 to 14-min duration at a selected time within the trialwindow. This particular timewas chosen by the test directorsand was not made available to the instrument operators.Thus, the collector/detection system was cycled in a contin-uous manner during a trial.

The series of trials spanned a time period of approxi-mately 4 weeks. All trials took place during the nighttime,because during the daytime the effect of the sun on the windcurrents caused greater unpredictability relative to that atnight. Thus, a successful execution of a bacterial aerosolrelease from the point of dissemination to the Py-GC/IMSanalytical detection device, which spanned approximately800 m, significantly relied on a steady current of wind.

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FIG. 4. Photograph of the quartz tube–microfiber filter aerosol-sample introduction region. Refer to the Experimental section for the legend.

A total of 27 trials was performed. The aerosol trials con-sisted of gram-negative EH bacterial particles and selected,representative trials along with a bacterial standard will bepresented.

Erwinia herbicola Standard

Figure 6 shows a contour plot of a GC/IMS data domainobtained by pyrolysis of EH, where the intensity parameteris the third dimension (off the page). This Py-GC/IMS ex-ample of an EH aerosol was collected from an outdoor trial,and prior to the trial it was known to the operator that arelatively high concentration of EH bacteria would be re-leased. The discontinuous peak at 4.25� 0.22 ms is thereactant ion peak (RIP), which consists of protonated watermolecule clusters. Proton transfer from water molecules toanalyte species effects the sample ionization. When the pro-ton affinity of a compound is greater than that of protonatedwater, the latter transfers the proton to the compound.62–64

Two phenomena happen: (1) a peak appears representativeof the compound, and (2) a depletion of the RIP occurs atthe same GC retention time as that of the compound. The

feature at 5.10� 0.20-ms drift time represents pyrolyzatespecies of the bacteria, and they elute from the GC columnfrom 6.00 to 8.06 s (dotted box). There is a difference of1.00� 0.038 ms between the drift times of the RIP and theEH pyrolyzate species. Unlike the definitive analysis of thepyrolysis ofBacillus subtilisvar. globigii (BG), where pi-colinic acid was identified as a discrete peak in the GC/IMSdata space,58–60 the peak used for EH representation (dottedbox in Figure 6) has not been identified.

Trial 14

Figure 7 presents aerosol and agar plate growth analysesof the temporal presence of EH. The Met-One total particlecount (PLA) over the 40-min trial window shows a majoraerosol event centered about 22:26:00. The agar-plategrowth analysis of the EH aerosol event (ACPLA) shows apeak that is in the same time frame as that of the PLA peak.Note that the aerosol event consists of approximately 300PLA, but only about 30 ACPLA is contained in the aerosolcloud. Thus, the cloud contains only 10% bacterial particles,and the remaining 90% can be characterized as nonbiolog-


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FIG. 5. Diagram of the timing for the three phases of a typical aerosol concentrator-Py-GC/IMS cycle.

ical particulate, dust, and inorganic debris, possibly origi-nating from the wheels of the moving aerosol generationvehicle. The Py-GC/IMS information is contained in eachsuccessive cycle, and these are found as boxes below thegraph. The tick mark above each box separates the aerosolcollection phase (to the left of the tick mark) and the pyrol-ysis and GC analysis phases (to the right of the tick mark).Thus each box represents a Py-GC/IMS interrogation cycle.The GC/IMS analysis phase in each cycle has no interro-gation of the ambient vapors/aerosols, because the aerosolcollector is turned off.

The pyrolysis of ambient particulates produces productsthat are observed in a GC/IMS data window for each cycle.The upper panel of Figure 8 shows the data window of fourcycles centered about the aerosol event in Figure 7. Cycle 9shows only a continuous RIP signal, and cycle 10 providesa signal that represents relatively low mass compounds fromthe particulate pyrolyzate as well as a depletion of the RIP.GC elution of a distinct set of compounds occurs at a reten-tion time between 6.53 and 8.27 s, and a drift time of5.55� 0.20 ms. Both the RIP and bacterial pyrolyzate spe-

cies experience a shift to slower drift times with respect tothose in Figure 6. This is because the pressure was increasedin the IMS cell.64 The increase in pressure was caused by afaulty air circulation pump in the IMS unit. However, thedifference in drift times between the RIP and pyrolyzate spe-cies is 0.95� 0.038 ms, which is very close to the 1.00-msvalue of the EH standard (Figure 6). This region describesthe same as that of the standard (Figure 6), and this is high-lighted by a dotted box in cycle 10, Figure 8. At test, with16 degrees of freedom, indicates that the drift time of thesample analyte peak and the RIP are linearly correlated(P � 0.001). Cycle 11 shows a relatively lower amount ofspectral features in the same GC/IMS data window (dottedbox). Cycle 12 shows essentially no pyrolyzate material andno RIP depletion. These observations in cycles 10 and 11are manifested as shaded areas in Figure 7. The overlap ofthe aerosol collection phases with the ACPLA graph (shadedbox areas in Figure 7) provides the pyrolyzate informationin GC/IMS data space. Keep in mind that the GC/IMS in-formation in a cycle happens after the aerosol collectionphase, and it is the latter that takes advantage of sampling

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FIG. 6. GC/IMS data window of a standard EH aerosol challenge to the XM-2 concentrator Py-GC/IMS system.

FIG. 7. Met-One aerosol (PLA) and ACPLA distributions and temporal disposition of the Py-GC/IMS cycles for Trial 14.


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FIG. 8. GC/IMS data windows for cycles 9–12 in Trial 14 and a phase analysis for each cycle with respect to the presence of bioaerosol.

the environment in a real-time manner with respect to theaerosol. A relatively greater RIP depletion is observed incycle 10 than in that of cycle 11 (Figure 8), because theshaded region for cycle 10 in Figure 7 encompasses a largerACPLA area than that of cycle 11.

The first cycle (cycle 10) in Trial 14, which shows a de-pletion of the RIP, represents the first presence of the EHaerosol, and the absence of EH from the sampled air can beascertained in the first blank cycle (cycle 12) after the lastcycle (cycle 11) displaying the presence of the pyrolyzatecompound.

Cycles 10–12 do not necessarily reflect the true times ofthe leading edge and trailing edge of the aerosol cloud withrespect to the Py-GC/IMS instrument. In order to investigatethis problem, Figure 8 provides an analysis of the phases ofcycles 9–12. In this trial, the aerosol collection and concen-tration phase of all four cycles constituted 120 s, althoughthe sample pyrolysis and processing phase time and the GCelution time for all four cycles were 28 and 50 s, respec-tively. The GC retention time (tR) of the EH pyrolyzate spe-cies (dotted box) is centered at approximately 7.4 s, andthese times are marked appropriately in the lower half ofFigure 8.

A time analysis of the leading edge of the EH aerosolcloud is found in the lower left side of Figure 8. The first

instance of the presence of EH is attR2 in cycle 10; thusbacteria must have been collected in the aerosol collectionphase of cycle 10. However, the leading edge of the cloudcould have occurred at a time between the beginning of thepyrolysis and processing phase in cycle 9 and near the endof the aerosol collection phase in cycle 10, and this regionis highlighted in Figure 8. The leading edge of the bacterialcloud could not have occurred during the aerosol collectionphase in cycle 9. Within the parameters of the Py-GC/IMSanalytical information in Figure 8, an exact time of arrivalof the leading edge of the bacterial aerosol cloud cannot beobtained. Thus, a time was chosen to represent the arrivaltime of the leading edge of the bacterial aerosol cloud, andthis is equal to the start of the aerosol collection phase ofthe first cycle yielding a biological signal in GC/IMS dataspace.

The trailing edge of the aerosol cloud can be treated inan analogous manner, and this analysis is presented in thelower right-hand side of Figure 8. The last instance for thepresence of EH is attR3 in cycle 11; thus, bacteria must havebeen collected in the aerosol collection phase of cycle 11.However, a blank cycle must follow a cycle displaying abacteria-derived signal in order to provide for a proper trail-ing-edge analysis. In this case, cycle 12 shows no biologicalsignal in Figure 8. The first instance of the absence of bio-

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logical cloud activity is at a GC retention time denoted bytR4. The absence of a signal attR4 indicates that no biologicalaerosol was collected in the aerosol collection phase in cycle12. Thus, the aerosol trailing edge could have occurred at atime after the beginning of the aerosol collection phase incycle 11 and no later than at the end of the GC elution phasein the same cycle. Because the exact time of the trailing edgeof the aerosol that passed over the Py-GC/IMS system couldnot be obtained, the time chosen to represent the trailingedge of the cloud was the beginning of cycle 12.

The analytical description of the aerosol event, the Py-GC/IMS mode of operation, and how the system interfacedand sampled the aerosol can be stated by the reasoning thatthe aerosol cloud is analog in nature and the method of sam-pling and analysis of the cloud is digital or segmented intime. Thus, the Py-GC/IMS device does not provide a con-tinuous, or seamless, analysis of the entire cloud. A discon-tinuous, yet constant interval, sampling by the system occursin real time, because the Py-GC/IMS only samples a portionof the time that the aerosol is present.

Figure 7 provides the experimental time versus PLAgraph with a superimposed graph of the ACPLA distributionfrom the agar plate, cultured aerosol data from the test di-rectors. Note that the ACPLA response, which shows base-line signal at the leading and trailing edges, occurs within

the same general time frame as the PLA peak. Only theaerosol collection phases of cycles 10 and 11 overlap in timewith the ACPLA aerosol distribution in Figure 7. It is thejuxtaposition of the time frame of the aerosol collectionphase in a cycle and the ACPLA aerosol distribution thatleads to the production of the indicator for EH presence inthe respective GC/IMS data window. This point reduces thedegree of importance that is placed on the presence/absenceof an aerosol event (PLA curve) as measured by a particlecounter. Thus, without an ACPLA-cultured biological aero-sol determination, the presence/absence of a bacterial aerosolcloud is predicated on the presence/absence of the selectpyrolyzate signal in GC/IMS data space in Figure 7, in con-trast to the presence/absence of an aerosol event in the Met-One aerosol particle count record.

The dotted-line graph in Figure 7 represents a plot of theaverage signal intensity, for all cycles in the trial, of thepyrolyzate feature in the outlined area in Figure 8. Thus, thisinformation is essentially a summary of the signal-to-noiseratio (S/N) of the EH biochemical analyte for the individualGC/IMS data-window plots for each cycle. Note that eachpoint in the S/N graph lies above the GC processing phaseof each cycle. One can observe the aerosol event on onegraph from a total particle (Met-One), bacterial particle (Pe-tri dish), and bacterial analyte signal (Py-GC/IMS) in Figure


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FIG. 10. GC/IMS data windows for cycles 4–7 in Trial 15.

7. Note that the most intense analyte signals (S/N curve)correlate where the aerosol collection phases of cycles 10and 11 overlap with the most intense peaks in the PLA andACPLA curves.

The 30 ACPLA that characterizes the EH aerosol cloudcan be converted to a maximum value of the number ofparticles that were collected onto the filter. The XM-2 aero-sol concentrator collects at a rate of 1000 L/min, and foreach cycle in a trial the aerosol concentrator collected for aperiod of 2 min. Separate experiments have shown that theefficiency of particle collection is 50 and 16% for 5- and 2-�m-diameter spheres, respectively. Thus, assuming that theEH cloud contained predominantly 5-�m size particles:1,000 L/min (0.5 efficiency) (2 min) (30 ACPLA)� 30,000EH-containing particles.

Trial 15

Figure 9 presents the PLA and ACPLA graphs of Trial15, and the trial window spanned�30 min. Figure 9 shows

a very low aerosol challenge of EH bacteria at approximately3 to 4 ACPLA. Cycles 5 and 7 appear to overlap with theACPLA aerosol distribution, with cycle 5 encompassing alarger area than that of cycle 7 (shaded areas). Figure 10shows the GC/IMS cycle data windows, and only cycle 5displays a GC/IMS signature (dotted box). The drift timedifference of the RIP and pyrolyzate species is 0.84 ms, andthe GC retention time spans peaks in the 6.67 to 8.00-srange. Apparently the second EH bacterial event centeredabout cycle 7 was too low an ambient aerosol challenge forthe Py-GC/IMS system. This aerosol event displayed amax-imum at 2 ACPLA. However, cycle 5 shows very good sen-sitivity for the system in general at an EH aerosol challengeof only 3 to 4 ACPLA. The S/N curve for the EH pyrolyzateanalyte in Figure 9 provides a satisfactory correlation withthe PLA and ACPLA curves. The total aerosol burden in thetime frame of cycle 5 is approximately 290 PLA; thus, thebiological particulate was present at a relative amount of 1.0to 1.4%. Assuming a predominance of 5�m-sized particles,

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�3000–4000 EH-containing particles were captured andpyrolyzed on the pyrolysis filter, which produced the pyro-lyzate species in cycle 5.

Trial 21

Figure 11 presents EH aerosol characterization results ofTrial 21. Two sharp events are observed in both the PLAand ACPLA graphs (Figure 12). The ACPLA graph providesa greater resolution of the biological portion of the aerosolevent than that of the PLA graph. The later biological aerosolevent in the ACPLA graph provides a relatively larger areathan that of the earlier event. Cycles 7 and 8 of the Py-GC/IMS analyses (Figure 12) provide an overlap of the respec-tive aerosol collection phases with each ACPLA event (dot-ted boxes). Figure 12 shows that cycle 6 and, however, cycle7 provide no GC/IMS data-window pyrolyzate information,whereas cycle 8 shows a noticeable presence of pyrolyzatespecies. The difference in drift time between the RIP andpyrolyzate species is 1.04 ms, and the pyrolyzate peaks spana retention time range from 5.14 to 7.07 s. Cycle 9 is similarto cycles 6 and 7; thus, the EH aerosol event in cycle 8 isbounded by the blank cycles 7 and 9. The area characterizingthe first biological aerosol event (cycle 7) in the ACPLAgraph in Figure 11 is significantly smaller than that in cycle8. This is most likely the reason that the presence of EHpyrolyzate species is not observed in cycle 7. Cycles 7 and

8 show the presence of EH bacteria from the ACPLA curve,and the S/N curve also mirrors this observation for pyroly-zate analyte. Excluding the very sharp peaks, the agar plategrowth results show an EH aerosol of approximately 6 AC-PLA. This value roughly translates into 6,000 EH-containingparticles collected onto the filter in cycle 8. With a totalaerosol particle count of approximately 200 ACPLA, theEH-containing particles constituted only 3% of the aerosolparticulates.

Conclusions

A relatively compact, two-dimensional analytical devicehas been presented for the detection of microorganism aer-osols in a western United States outdoor, desert environ-ment. In a stand-alone mode, the atmospheric-pressure-based Py-GC/IMS device relies on an aerosol concentratorfor the introduction of biological aerosol particulates. In arelatively short period of time, organism presence in the aircould be detected in amounts as low as 3–4 ACPLA by thePy-GC/IMS instrument after only 2 min of aerosol precon-centration and 0.5 min of processing.

Detection of presence of the gram-negative EH organismbioaerosol was effected by the presence of a distinct pyro-lyzate species in GC/IMS data space. However, the identityof the pyrolyzate species was not determined. The Py-GC/


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FIG. 12. GC/IMS data windows for cycles 6–9 in Trial 21.

IMS instrument was shown to serve as an effective comple-ment to a standard aerosol particle counter.

Acknowledgment

We wish to thank Alice I. Vickers for the preparation andediting of the manuscript.

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