Aerosol Science 36 (2005) 801–812

www.elsevier.com/locate/jaerosci

The sea surface microlayer as a source of viral and bacterialenrichment in marine aerosols

Josephine Y. Aller∗, Marina R. Kuznetsova, Christopher J. Jahns, Paul F. Kemp

Marine Sciences Research Center, Stony Brook University, Stony Brook, NY 11794-5000, USA

Received 26 April 2004; received in revised form 4 October 2004; accepted 28 October 2004

Abstract

Marine aerosols are formed primarily by the eruption of rising bubbles through the sea-surface microlayer (SML),and aerosol formation is the main vector for transport of bacteria and viruses across the air–sea interface. Theprocesses by which materials are transported to and through the SML to the atmosphere results in an enrichmentin the SML of microbial and other organisms. We evaluated concentrations of marine bacteria and viruses innatural aerosols and in those simulated by bubbling sea sprays and compared them to the concentrations in SML(200–400�m thick) and in subsurface water. Association of microorganisms with transparent gel-like organicparticles and physiological status of bacteria were also assessed. We found a 15–25-fold enrichment in bacteriaand viruses during transport from subsurface waters to the SML, and then into the atmosphere. The majority ofmicroorganisms in aerosols were found embedded in the organic particles. Large portions of microorganisms inthe SML were also associated with the particles, while in subsurface waters most of them were free-living. Alarger percentage of damaged and less active bacterial cells occur in the microlayer than in subsurface waters, andpreliminary data suggest that an even greater percentage occurs in aerosols. Our data support the idea that the SMLin a major source of microorganisms entering the atmosphere from water bodies. Aerosolization is potentially animportant long-distance dispersal mechanism and may account for observed cosmopolitan distributions of somebacteria.� 2004 Elsevier Ltd. All rights reserved.

Keywords:Bioaerosols; Sea surface microlayer; Bacteria; Viruses; Long-distance transport

∗ Corresponding author. Tel.: +1 631 632 8655; fax: +1 631 632 3066.E-mail address:jyaller@notes.cc.sunysb.edu(J.Y. Aller).

0021-8502/$ - see front matter� 2004 Elsevier Ltd. All rights reserved.doi:10.1016/j.jaerosci.2004.10.012

802 J.Y. Aller et al. / Aerosol Science 36 (2005) 801–812

1. Introduction

Bubbles generate the major portion of marine aerosols that are easily suspended and transported in thelower atmosphere (Woodcock, 1953; Gustafsson & Franzen, 2000; Grammatika & Zimmerman, 2001).Breaking waves and rain impacting the sea surface are major sources of bubbles in the upper ocean, asis boat traffic (Monahan, Fairall, Davidson, & Boyle, 1983; Klassen & Roberge, 1999). Bubbles can bedispersed to depths of several meters (Grammatika & Zimmerman, 2001). Dissolved surface-reactive in-organic and organic compounds, and small particles including bacteria and viruses collect on the surfaceof these bubbles. Bubbles eventually rise through the water column and upon reaching the sea surface,burst and eject aerosol droplets into the atmosphere, delivering the material carried by the bubbles to thesurface microlayer (SML) and to the atmosphere.

The sea SML is a unique biological, physical and chemical habitat at the interface between the at-mosphere and hydrosphere. Older reports suggest that bacteria may be 102–104 times more abundantin the SML than in subsurface waters (Sieburth, 1963; Bezdek & Carlucci, 1972; Sieburth et al., 1976),and it appears that microbial activity is elevated in the SML; e.g. extracellular enzyme activity is greatlyenhanced compared to subsurface waters (Kuznetsova & Lee, 2001). Dissolved and particulate organicmatter can be enriched up to 1000 times in the SML compared to subsurface waters (Hardy, 1982; Liss& Duce, 1997). Bubble scavenging and delivery to the SML is the likely mechanism for this enrich-ment.

Aerosols are further enriched in both bacteria and viruses in comparison to either the SML or subsurfacewaters. These aerosols include part of the organic material, metals, bacteria and viruses collected bybubbles (Blanchard, 1975; Blanchard & Syzdek, 1982). Enrichment in marine aerosols can be very highrelative to subsurface waters (up to three orders of magnitude in some locations,Marks, Kruczalak,Jankowska, & Michalska, 2001).

Aerosol formation is the main vector for transport of bacteria and viruses across the air–sea interface(e.g.Matthais-Maser & Jaenicke, 1994; Posfai, Li,Anderson, & Buseck, 2003), and aerosols can transportmicrobes for long distances. Early work suggests that bacterial endospores and fungal spores may travelover intercontinental distances (e.g.Bovallius, Roffey, & Henningson, 1980). Non-marine aerosols fromAfrica cross the tropical Atlantic Ocean carrying particulates, microorganisms and other contaminantsinto the Caribbean region (De la Mota, Betancourt, Detres, & Armstrong, 2003; Detres & Armstrong,2003). Several studies have shown that marine aerosols large enough to contain microbes and organicparticles such as viruses (e.g. 1–20�m) can be transported for hundreds of kilometers from their source(e.g.Klassen & Roberge, 1999; Moorthy, Satheesh, & Krishna Murthy, 1998; Chow et al., 2000), andremain liquid, provided that the relative humidity is high enough (over∼ 70%). Aerosols of this sizemay remain suspended in the atmosphere for weeks (e.g. 31 days residence time,Dueñas, Fernandez,Carretero, Liger, & Cañete, 2004). There is evidence that at least some marine bacteria remain viable afteroverland transport in aerosols (200 m inland, collected at 30 m above sea level;Marks et al., 2001), de-spite exposure to ultraviolet radiation, changes in temperature and salinity, and other immediate stresses.These data were based on culture plating, and because only a small fraction of viable marine bacteria willgrow on culture plates, it is very likely that the actual number of viable bacteria in aerosols was greatlyunderestimated.

Here, we describe a 4-month study of bacterial and viral abundance, and bacterial activity in marineaerosols, SML and subsurface waters of Long Island Sound, a northeast US estuary with 8 millionpeople living in its watershed. Our data support a conclusion that aerosol formation and transport in

J.Y. Aller et al. / Aerosol Science 36 (2005) 801–812 803

the atmosphere is an important mechanism for the dispersal and biogeography of marine microbes, andmay account for the maintenance of cosmopolitan distributions of some marine bacteria. Many microbialpathogens survive drying in non-marine aerosols, and their dispersal in the atmosphere may present asubstantial hazard to public health (Carducci, Gemelli, Cantiani, Casini, & Rovini, 1999; Brandi, Sisti,& Amagliani, 2000; Alexandersen, Alexandersen, Brotherhood, & Donaldson, 2002). We believe thattransport of viable microbes in marine aerosols may present a substantial human health risk, particularlyin coastal metropolitan areas.

2. Materials and methods

2.1. Field sampling locations

Over a 4-month period (June–September 2003) subsurface water, SML and aerosol samples werecollected from locations adjacent to Long Island, New York. Samples of the subsurface water, SMLand aerosols were collected during in situ experiments using a small floating catamaran in Stony BrookHarbor. Samples of aerosols were collected from the R/V Seawolf in a 200 km transect from the conti-nental shelf off Montauk Pt. (eastern end of Long Island) to the shelf break (∼ 250 km); and from theBridgeport/Port Jefferson Ferry P.T. Barnum in transects across the central portion of Long Island Sound(Table 1).

Meteorological conditions varied between sampling times and with location (i.e. open Sound, offshoreShelf, or at the shoreline). We tried to be consistent during simulated aerosol collections with the floatingcatamaran. Samples were collected on sunny days between 1000 and 1600 h with air temperatures rangingin 25± 3◦C and south to southeast winds at∼ 3–4 m s−1.

Table 1Source and type of sample collected

Collection platform Sampling location Type of sample collecteda Sampling dates

Small floating Stony Brook Harbor Aerosol Microlayer Subsurface 7-June, 17-JuneCatamaran samples samples water 23-June, 25-June

7-July, 15-July24-September

R/V Seawolf Long Island Sound and Aerosol samples Subsurface water 14–17 July dailycontinental Shelf waters

Ferry Central Long Island Sound Aerosol samples Subsurface water 10-June, 19-June,26-June

Laboratory beaker Stony Brook Aerosol samples Bulk water 14-FebruaryHarbor water 12-March

16-June17-July

aAll samples were collected in replicate.

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2.2. Ambient marine aerosol sampling

Aerosol samples were collected on Pallflex Tissuequartz filters at the end of a PVC pipe that extended1.5–3 m away from the ship, at a rate of 11 L min−1 of air. Collection times averaged 50 min from theFerry with collectors held 3–4 m above the sea surface, and 6 h from the RV/Seawolf with collectors held4 m above sea surface. Collectors positioned toward the side of the vessel effectively collected aerosolsproduced by bubbles generated in the ship’s wake. Filters removed from holders were immediately placedinto sterile artificial seawater with 2.5% buffered formaldehyde and refrigerated. Further details can befound in Kuznetsova, Lee, and Aller (submitted).

2.3. In situ generation and collection of aerosols

Aerosols were generated in situ using a sintered glass filter bubbling source (pore size 15–40�m)positioned∼ 15 cm under the water surface and driven by a battery-operated pump. Aerosols werecollected at 10 cm above the water surface on a set of inverted funnels covered with triple boiled acid-washed 1-�m nylon mesh filters using a high flow vacuum pump.The generation and collection system wasclamped to a small floating catamaran that did not restrict water flow under the funnels (see Kuznetsovaet al., submitted for more details). The size spectrum of bubbles produced by the bubbling source closelysimulates that generated beneath a breaking wave (Grammatika & Zimmerman, 2001; Cloke, McKay, &Liss, 1991). The size spectrum of droplets generated as the bubbles burst at the water surface approximatedthe range of droplets (1.2–20�m diameter) comprising most of the mass of natural marine aerosols(Seinfeld & Pandis, 1998). Collection lasted for 1–2 h, after which each filter was removed and cut intohalf. One-half was immediately placed into artificial seawater with 2.5% formaldehyde for bacteria andvirus (as virus-like particles (VLPs)) counts. The other half was placed in distilled water to determinethe mass of salt as a proxy for the liquid volume of the collected aerosol. Laboratory simulations withthe same bubbling unit showed no significant difference(P > 0.05) in the salinity of the aerosol at themoment of generation, and ambient seawater salinity. Aerosol production increased the concentrationof salt particles in the air at least 20-fold, and we are confident that the aerosols generated by bubblinggreatly exceeded the concentration of natural marine and terrestrial aerosols per volume of air; however,we did not actually measure the concentration of non-salt-bearing terrestrial aerosols (Fig. 1).

2.4. Laboratory experiments

Aerosol simulation experiments were carried out in the laboratory in a 6-l beaker with seawater freshlycollected from Stony Brook Harbor and a bubble source as described above. The liquid aerosols werecollected on an impaction plate positioned 1–2 cm above the water surface.

2.5. Sea SML and subsurface water sampling

Microlayer samples (∼ 200–400�m thick) were obtained using a polyester 1-mm mesh screen (afterGarrett, 1965; Kuznetsova & Lee, 2001). Subsurface water samples were collected in the same locationsby submerging a polypropylene bottle by gloved hand 15 cm(±2 cm) below the sea surface. The bottlewas opened and closed underwater to minimize SML interference. The total volume of the surface layermaterial potentially introduced into the sample by the glove and the bottle surfaces that pass the air–water

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estuaryinner shelf

inner shelf after stormshelf break

0

40

80

120

160C

ells

lite

r of

air

-1bacteriaVLPs

boat wake0

500

1000

1500

2000(a) (b)

Fig. 1. Comparison of bacterial and viral (VLPs) concentrations in natural aerosols collected from 4 m above the sea surfaceunder varying environmental conditions (a) off the bow of ship and (b) to the side of the ship’s wake.

boundary was negligible, because the surfaces got rinsed quickly in the dynamic subsurface environment,and the total volume introduced into the bulk material was minor compared to the surrounding bulkwater volume. Samples were preserved immediately with 2.5% formaldehyde and kept on ice in the dark(10–15 min) during transportation to the laboratory.

2.6. Abundances of Bacteria, VLPs, and transparent exopolymer particles

Bacterial,VLP, and semi-transparent particle counts were made on duplicate slides using a 100× NikonEclipse E400 epifluorescence microscope with a MetaMorph� Imaging System. A minimum of 100 cellsper slide were counted for each sample for an estimated precision±5%. Bacterial abundances weredetermined using the fluorescent dye 4′, 6-diamidino-2-phenylindole (DAPI) (afterPorter & Feig, 1980).VLP counts were made using SYBR Gold, a nucleic acid stain that detects double or single strandedDNA or RNA (Molecular Probes, Inc.) using a modification of published procedures (Hewson, O’Neil,Fuhrman, & Dennison, 2001).

Slides were generally prepared immediately upon return of samples to the laboratory, however thiswas not always possible. The effect of sample storage was experimentally examined. Counts of VLPs inpreserved water samples were found not to vary significantly(P > 0.05) if counted within 2 weeks ofsample collection. There were no significant changes(P > 0.05) in counts between the slides prepared andcounted immediately after sampling and ones prepared immediately, but held in the dark and refrigeratedfor more than 6 weeks before counting. Counts of bacteria also showed no significant changes(P > 0.05)over a 4-week time period in refrigerated preserved water and sediment samples. Transparent exopolymerparticles (TEP) were quantified microscopically after staining with Alcian Blue dye (polysaccharide-rich particles,Passow & Alldredge, 1994), or Coomassie Blue dye (proteinaceous particles,Berman &Viner-Mozzini, 2001).

2.7. Damaged and metabolically active bacteria

Cells with damaged membranes were detected by staining with Propidium Iodide and enumerated rela-tive to total cells stained by DAPI (Joux & Lebaron, 2000). rRNA-targeted fluorescent in situ hybridization(FISH) was carried out with Alexa-532 labeled oligonucleotide probes ALF-1b to�-proteobacteria and

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EUB338 for Eubacteria using a modification of published methods (Kemp, Lee, & LaRoche, 1993; Kemp,1995; Howard-Jones, Frischer, &Verity, 2001), including dual staining with DAPI. Cells having sufficientrRNA to yield visible fluorescence were enumerated relative to total DAPI-positive cells.

3. Results

3.1. The net bacterial and viral flux from water to aerosols

At any given time the net bacterial and viral fluxes from the ocean to the atmosphere is equal to theaverage concentration of bacteria or viruses within the aerosol, times the total aerosol production. Thetotal marine aerosol production can be evaluated directly by measuring salt concentrations on aerosoltraps. Salt enrichment in the microlayer does not usually exceed 3% (Liss & Duce, 1997) so that aerosolsalt content reflects the amount of seawater that produced the aerosol. The enrichment factor is definedas the ratio of the average concentration of bacteria or viruses in two different sample sources (e.g.microlayer compared with bulk seawater) with a correction for any difference in salinity or relative tosalinity.

The average concentrations of bacteria and VLPs on 6 sampling dates from mid-June to the end ofSeptember 2003 were substantially elevated in aerosols compared to subsurface waters (Fig. 2). Comparedwith subsurface waters, bacteria were enriched 10-fold on average in aerosols (enrichment is significantat P < 0.05; maximum enrichment was 22-fold), and 6-fold on average in the SML (maximum 10-foldenrichment). Viruses were enriched on average 10-fold in aerosols (maximum 15-fold) and 7-fold in theSML (maximum 18-fold).

Variations in surface water to SML (Fig. 3a and b), surface waters to aerosol, and between the SMLand aerosols (Fig. 3c) likely reflect temporal differences in environmental conditions including windspeed and direction, air and water temperature, air humidity, and biochemical parameters such as DOMconcentrations. We know of no other measurements of enrichment for bacteria or viruses that are nativeto seawater and believe that it may be useful for initial ecological hazard assessments when combinedwith knowledge of the concentration of microorganisms in subsurface waters and salt aerosol flux.

June - September0

5

10

15

20

25

Bac

teria

106

ml-1

Stony Brook Harborsubsurface watermicrolayeraerosol

June-September

0

5

10

15

20

25

VLP

s 10

6 m

l-1

Fig. 2. Bacteria and viral (VLPs) abundances collected from replicate Long Island Sound subsurface water, microlayer, andaerosols samples averaged over 6 sampling times from mid-June to late-September, 2003.

J.Y. Aller et al. / Aerosol Science 36 (2005) 801–812 807

0 5 10 15 20

cells 106 ml-1 in subsurface waters

0

5

10

15

20

25

30

35

40

cells

106

ml-1

Bacteriamicrolayeraerosol

(a)0 5 10 15 20

VLPs 106 ml-1 in subsurface waters

0

5

10

15

20

25

30

35

40

VLP

s 10

6 m

l-1

VLPsmicrolayeraerosol

(b)

0 5 10 15 20 25 30 35 40

cells or VLPs 106 ml-1 in SML

0

5

10

15

20

25

30

35

40

cells

or

VLP

s 10

6 ml-1

in a

eros

ols

SML-Aerosol EnrichmentVLPsbacteria

(c) SS-SM

LSS-A

eros

olSM

L-ae

roso

l

0

5

10

15

20

25

Enr

ichm

ent F

acto

r

BACTERIAVLPs

(d)

Fig. 3. Enrichment of bacteria (a) and viruses (VLPs) (b) was apparent in the sea SML and marine aerosol compared withsubsurface waters in replicate samples collected on 6 occasions between 17-June and 24-September, 2003 in Stony BrookHarbor. (c) Enrichment of bacteria and viruses (VLPs) is also evident in these samples when aerosols are compared with the seaSML. (d) Calculated enrichment factors (EFs) between the SML and subsurface waters (SS), aerosols and SS, and aerosols andthe SML averaged 4–11 for both bacteria and VLPs.

3.2. Particle-associated microorganisms

The upper ocean contains suspended transparent exopolymer particles (TEP), comprised of polysac-charide exudates by phytoplankton (Mopper et al., 1995; Passow, 2002), proteinaceous particles (Long &Azam, 1996; Berman & Viner-Mozzini, 2001), and amorphous DAPI-stained particles (Mostajir, Dolan,& Rassoulzadegan, 1995; Simon, Gorssart, Schweitzer, & Ploug, 2002), all of which are generally colo-nized by microorganisms. In our aerosol samples we observed numerous DAPI-stained particles, as wellas TEP and proteinaceous particles. All the three particle types resembling folded sheets or misshapenglobules and varying in size from 3 to about 40�m were similarly enriched in the microlayer (∼ 2–5-fold)and aerosols (20–40-fold). We tested the idea if the association of microorganisms with such particlescould be significant in laboratory experiments in which aerosols were generated artificially. In subsurfacewater, bacteria and viruses were predominantly free-living, with only 11±4% of all bacteria and 17±12%VLPs associated with particles. In the SML 23±1% of bacteria and 15±1% ofVLPs were associated withparticles, and in aerosols, 59±3% of bacteria and 35±11% ofVLPs were associated with particles (Fig. 4).

3.3. Metabolic activity of bacteria

We found that more cells had compromised membranes in the microlayer compared with subsurfacewaters, and relatively fewer cells contained enough rRNA to be detected by FISH (Fig. 5a). Active cells

808 J.Y. Aller et al. / Aerosol Science 36 (2005) 801–812

Fig. 4. Transparent gel-like organic particles with embedded bacteria and VLPs from aerosol sample collected on July 7, 2003.

subsurface microlayer0

2

4

6

Bac

teria

106

ml-1

totala-proteobacteria

Eubacteriamembrance compromised

microlayer

0.15 m

1 m

15 m

30 m

0 25 50 75 100

% α-Proteobacteria ofDAPI count

(a) (b)

Wat

er D

epth

(m

)

Fig. 5. Comparison of the physiological state of bacteria in subsurface and microlayer samples averaged over 6 sampling timesin SB Harbor between 17-June and 24-September 2003. (a) Total cell counts were on average twice as high in microlayersamples, however; the fraction of EUB338 and ALF1b probe-binding cells relative to DAPI-stained cells were 2–4 times greaterin subsurface waters and propidum idodide stained cells were 5 times lower, demonstrating a greater proportion of active cellswith intact membranes than in the microlayer. (b) Depth profile of ALF1b FISH-detectable cells relative to DAPI-stained cellsaveraged for 5 weekly sampling dates from 7-July to 4-August in Long Island Sounds illustrates the severe drop in bacterialactivity between subsurface waters and the microlayer.

(those labeled by FISH) declined from over 60% in subsurface waters to 8% in the microlayer (Fig. 5b),suggesting that inactive cells are preferentially scavenged or that cells stop growing when delivered tothe microlayer. On the other hand, there was considerable enrichment of VLPs from subsurface water

J.Y. Aller et al. / Aerosol Science 36 (2005) 801–812 809

to the microlayer. We have no means to determine whether these were produced in the microlayer, orscavenged and delivered to the microlayer.

Rather than nutrient-rich, stable and nurturing environments, microlayers appear to be habitats wherephyto- and zooneuston concentrate along with anthropogenic poisons (Hardy, 1982; Liss & Duce, 1997).Viral abundances are enhanced and the potential for grazing and viral infection are great. A variety ofphysical factors including UV radiation, temperature and wind can occur in extremes, and variations inevaporation and precipitation create exceptionally stressful conditions. Such stresses may account forour observations of an increased proportion of membrane-damaged cells, and a decreased proportion ofactive cells in the SML.

4. Discussion

Many microbial pathogens survive drying in aerosols, and their dispersion in the atmosphere maypresent a hazard to public health (Carducci et al., 1999; Brandi et al., 2000; Alexandersen et al., 2002).Aerosols that contain pathogens promote infection by facilitating inhalation, ingestion and contact withthose pathogens (Gregory, 1972). Although most seawater microorganisms are harmless to mammals andhigher plants (Joint, 1995), hazardous species can be present in seawater as a result of sewage inputsthat contain a spectrum of human-borne pathogens (Mitchell, 1971). Ponds contaminated by farmingactivities or livestock wastes can flood waterways that empty into coastal areas and thus also introducepathogenic microorganisms into seawater (Mitchell, 1971). Recent public warnings concern seaports aspotential targets for terrorist attacks coupled with the possible use of biological weapons (Amass & Clark,1999; Peters, 2002) make this assessment particularly relevant.

Over the past decade, a substantial research effort has been directed toward identifying pathogensin seawater (Lewis, 1995; Baker & Bovard, 1996; Bronze, Huycke, Machado, Voskuhl, & Greenfield,2002) and toward determining survival of viruses and bacteria and preservation of their infectivity in theairborne phase (Hyslop, 1979; Cole & Cook, 1998; Kong, Lee, Law, Law, & Wu, 2002). We have shownthat bacteria were enriched∼ 6-fold from subsurface waters to the sea SML water, but were up to 22-foldenriched in marine aerosols compared to bulk seawater. Viruses were enriched on average 7-fold in themicrolayer, but were enriched in aerosols up to 15-fold. These substantial enrichments must be consideredin any evaluation of health risks associated with natural marine aerosols. Since most marine viruses havephysical properties similar to other known viruses (Steward, Montiel, & Azam, 2000) measurement ofnatural viral fluxes could be useful in future predictions of pathogen transport.

Viability of marine bacteria and viruses in aerosols is considerably challenged. Aerosol droplets inthe atmosphere undergo substantial temperature and salinity changes, or, ultimately, dry out. The mi-croorganisms carried by aerosols are exposed to higher doses of UV light than in bulk water. However,the fact that the majority of bacteria and viruses appear to be embedded in transparent gel-like organicparticles in the microlayer and in aerosols, may provide some degree of physical protection from UVradiation and drying as well as a nutrient source allowing some to remain viable for extended periodsin the atmosphere. At the same time, it is apparent that a greater percentage of damaged and less activebacterial cells occur in the microlayer and based on preliminary data, possibly an even greater percentageoccur in aerosols. Aerosol formation and transport in the atmosphere is clearly an important mechanismfor the dispersal of microbes many of which have been shown to have worldwide distributions (Mullins,Britschgi, Krest, & Giovannoni, 1995; López-Garcia, López-López, Moreira, & Rodriguez-Valera, 2001)

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and that transport of viable microbes in marine aerosols may present a substantial hazard to public healthparticularly in coastal regions. If airborne bacteria can retain their viability, they may help to account forthe maintenance of their cosmopolitan distributions.

Acknowledgements

We thank S. Cluett, captain of the RV/Seawolf and Fred Hall and the Bridgeport/Port Jefferson Steam-boat Company for facilitating sample collection. K.S. Chiang and M.Z. Amberger assisted with samplingand sample workup. M.M. Dantzler provided laboratory assistance. We thank C. Lee for constructivediscussions. Support from the NSF Grants OCE-9820277 to J.Y. Aller and OCE-0117208 to C. Lee madethis study possible. MSRC contribution No. 1276.

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