raman spectroscopic and laser scanning confocal microscopic analysis of sulfur in living...

Ž .Chemical Geology 180 2001 3–18www.elsevier.comrlocaterchemgeo

Raman spectroscopic and laser scanning confocal microscopicanalysis of sulfur in living sulfur-precipitating marine bacteria

Jill Dill Pasteris a,), John J. Freeman a, Shana K. Goffredi b, Kurt R. Buck b

a Department of Earth and Planetary Sciences, Washington UniÕersity, Campus Box 1169, St. Louis, MO 63130-4899, USAb Monterey Bay Aquarium Research Institute, P.O. Box 628, Moss Landing, CA 95039, USA

Abstract

Laser Raman microprobe spectroscopy and laser scanning confocal microscopy were used to determine the presence andspeciation of sulfur in sulfur-oxidizing, marine bacteria from Monterey Bay, CA. The bacteria studied include: large,filamentous Thioploca and Beggiatoa, endosymbionts in the vesicomyid clam Calyptogena kilmeri, and a filamentousbacterium of undetermined species. All of these bacteria were shown spectroscopically to store elemental sulfur insubmicrometer to several micrometer diameter vesicles. More detailed Raman spectroscopic study of the vesicles inThioploca and Beggiatoa provided further chemical and structural characterization of the elemental sulfur. The sulfur isbonded in the common, stable S ring configuration and is of an extremely fine-grained microcrystalline form. No additional8Ž .organo sulfur compounds were detected spectroscopically in the vesicles under the low laser powers required to preservethe molecular structure of the sulfur. The present spectroscopic and optical data stand in contrast to reports and inferences ofliquid-like elemental sulfur or homogeneous, complex sulfur compounds in other sulfur-oxidizing bacteria. The findings ofthis study are compatible with a model of sulfur vesicles as dominated by microcrystalline solid elemental sulfur, perhapsembedded in a matrix andror confining membrane of organic material. The high reactivity and solubility observed in thesevesicles is attributed to the extremely fine grain size of the solid elemental sulfur. q 2001 Elsevier Science B.V. All rightsreserved.

Keywords: Bacteria; Sulfur; Vesicle; Raman spectroscopy; Thioploca; Beggiatoa

1. Introduction

Chemoautotrophic sulfur-oxidizing bacteria usethe energy derived from the oxidation of hydrogen

Žsulfide, bisulfite, or thiosulfate using either oxygen.or nitrate as the electron acceptor to fix carbon

Ž .Schlegel and Bowien, 1989 . Sulfur-oxidizing bac-teria may exist as free-living large filaments or sin-

) Corresponding author. Fax: q1-314-935-7361.Ž .E-mail address: [email protected] J.D. Pasteris .

Žgle cells e.g., Thioploca, Beggiatoa, Thiomargarita,. ŽThiobacillus spp., Thiomicrospira spp. e.g., Nelson

and Hagen, 1995; Fossing et al., 1995; Schulz et al.,. Ž1999 , endosymbionts in metazoans e.g., Vetter,

.1985; Nelson and Fisher, 1995 , or epibionts onŽ . Ž .protists Buck et al., 2000 or metazoans Ott, 1996 .

All these bacteria share environments characterizedby elevated levels of hydrogen sulfide produced bybacterial sulfate reduction in sediments, e.g., hy-drothermal vents, cold seeps, or silled anoxic basins.Sulfur-oxidizing bacteria are thought to be importantto global biogeochemical processes due to their

0009-2541r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved.Ž .PII: S0009-2541 01 00302-3

( )J.D. Pasteris et al.rChemical Geology 180 2001 3–184

Ž .widespread distribution, their in some cases highbiomass and their coupled role in sulfur and nitrogen

Ž .cycles Fossing et al., 1995; Otte et al., 1999 . Anultrastructural feature shared by all sulfur-oxidizingbacteria is the presence of membrane-bounded spher-ical to subspherical vesicles. These vesicles are

Žthought to be sites of sulfur storage Vetter, 1985;.Steudel, 1989 .

Numerous questions remain about the nature ofthe sulfur in these marine bacteria. The bacteriathemselves can range down to micrometer scale, andthe sulfur vesicles may be even smaller. The mostbasic question is whether the observed vesicles in-deed contain sulfur. Due to the small volumes ofmaterial within putative sulfur vesicles, however, itis difficult to distinguish them from other vesicle-like

Žstorage structures, such as PHB poly-b-hydroxy-.butyrate bodies. Other properties of sulfur and its

compounds, for instance, high vapor pressure andlow melting point, hamper analysis. Sulfur is knownfor its instability under an electron beam, particu-larly, while under vacuum. These factors make itdifficult to document the presence of sulfur in bacte-ria and, particularly, to identify exactly where thesulfur resides. In addition, questions remain concern-ing the chemical speciation of the sulfur, i.e., whether

Ž .it occurs as elemental sulfur in chains or rings or asŽ .sulfur-bearing organic compounds, such as poly-

Žthionates or organic polysulfides Steudel, 1989;.Prange et al., 1999 . Although the diversity of

Žsulfur-oxidizing bacteria is vast Kelly, 1989; Nel-.son, 1989 , the actual presence and nature of sulfur

have been investigated in detail only for a fewŽorganisms, notably Chromatium Hageage et al.,

. Ž .1970 , Beggiatoa Lawry et al., 1981 , purple andgreen sulfur-oxidizing bacteria from the familiesChromatiaceae, Ectothiorhodospiraceae and Chloro-

Ž .biaceae Prange et al., 1999 , and endosymbionts ofŽone lucinid and one vesicomyid clam species Vetter,

.1985 . It currently is assumed that all other sulfur-oxidizing bacteria, both free-living and symbiotic,have similar mechanisms of processing the sulfur

Žgenerated by chemosynthesis with the apparent ex-ception of sulfur-filamentous bacteria described by

Ž . Ž ..Taylor and Wirsen 1997 and Taylor et al. 1999 .Techniques, such as transmission electron mi-

Ž . Ž .croscopy TEM , X-ray diffractometry XRD , elec-Ž .tron microprobe analysis EMPA and X-ray absorp-

Ž .tion near-edge spectroscopy XANES , have beenapplied to the sulfur vesicles in some bacteriaŽHageage et al., 1970; Nicolson and Schmidt, 1971;Lawry et al., 1981; Vetter, 1985; Prange et al.,

.1999 . TEM can provide excellent documentation oforganic and inorganic morphology at the submicrom-

Ž .eter level Fig. 1 . However, the technique calls forcomplex sample preparation in order to maintain thesample’s morphology and to permit the necessary

Ž .thinning of the material Maier et al., 1990 . Unfor-tunately, elemental sulfur readily dissolves in sol-vents used during the dehydration typical of tissue

Žprocessing e.g., Strohl et al., 1981; Vetter, 1985;Ž . Ž ..this paper, Fig. 1 B , D . The sulfur that remains is

subject to thermal degradation under the electronbeam. TEM photomicrographs of known sulfur-pre-cipitating bacteria, such as Thioploca and Beggia-toa, typically show minute transparent features thatmay be interpreted as Asulfur drop-outB regions andsmaller electron-dense features that may be inter-

Žpreted as residual sulfur Maier et al., 1990; Vetter,.1985; this paper, Fig. 1 . For samples in which the

sulfur vesicles can be stabilized, selected-area elec-tron diffraction techniques offer the ability to under-stand any crystalline structures of sulfur in individualvesicles. To our knowledge, no such analyses havebeen undertaken.

Many of the sample-handling and sample-stabilityproblems of TEM also hinder EMPA of sulfur-bearing vesicles. EMPA has micrometer-scale spatialresolution and can provide qualitative or quantitativeanalysis of the elemental composition of the vesicles.

Ž .However, both 1 detection of organic compoundsŽi.e., recognizing atomically light elements, such as

. Ž .C, H, N and O, in addition to AheavyB S , and 2distinguishing the presence of organic compoundsfrom the simple physical intergrowth of elementalsulfur with some organic material are very difficultwith an electron microprobe. Thus, Lawry et al.Ž . Ž .1981 and Vetter 1985 identified by EMPA thedominance of element sulfur in B. alba vesicles andin symbionts in the gills of Lucinoma annulata,respectively; however, they could not rule out theadditional presence of organosulfur compounds.

In contrast to the chemical analytical capabilitiesof EMPA, X-ray diffraction provides an excellentmeans for documenting the specific crystalline struc-ture of a material, thereby allowing for identifica-

( )J.D. Pasteris et al.rChemical Geology 180 2001 3–18 5

Ž .Fig. 1. Electron micrographs of chemoautotrophic sulfur-oxidizing bacteria. A Scanning electron micrograph of critical-point-driedŽ . Ž . Ž .Thioploca trichomes T inside a common polysaccharide sheath sh . Scale bars100 mm. B Transmission electron micrograph of a

Ž . Ž . Ž .single Thioploca trichome illustrating the cell wall arrowhead , the septum arrow separating two adjoining cells, the large vacuoles VŽ .that comprise most of the cell volume, and the sulfur vesicles S . Vesicles out of which the sulfur has been dissolved during the

Ž .dehydration series appear bright white. Scale bars10 mm. C Transmission electron micrograph of the gill of C. kilmeri illustrating theŽ . Ž . Ž .bacteriocytes B , one of which dominates the field of view, and the peripheral microvilli M . Scale bars10 mm. D Transmission

Ž . Ž Ž ..electron micrograph of the gill of C. kilmeri illustrating the individual bacteria b within a bacteriocyte see C . The individual bacteriaŽ .are complex, rounded, white and gray structures containing bright white empty sulfur vesicles S . Scale bars1 mm.

tion. However, XRD is only sensitive to crystallinespecies, and the sample is subject to possible X-ray-induced degradation during analysis. In addition,sufficient sample material must be collected to en-able detection. This type of AbulkB analysis does notyield direct information on the spatial distribution ofsulfur within the bacteria. XANES, as implemented

Ž .by Prange et al. 1999 through application of syn-

chrotron radiation, not only can provide structural-chemical information on the absorbing atoms, butalso can be applied to intact bacteria with theirassociated sulfur spherules. However, beam intensi-ties on the spherules are very high; interpretation ofthe spectra in terms of actual sulfur species is com-plex and typically proceeds by comparisonranalogywith spectra of known standards.


To avoid the limitations and difficulties of theabove analytical techniques, we selected a combina-tion of optical microscopy, particularly, laser scan-ning optical confocal microscopy, and laser Ramanmicroprobe analysis to locate and determine the spe-ciation of sulfur vesicles in four types of sulfur-oxidizing bacteria. There are several advantages tothese two approaches. The optical confocal micro-scope, employing a combination of epireflection andautofluorescence imaging, can be used to determine

Žthe 3-D spatial distribution of highly refractile i.e.,.showing high optical relief sulfur precipitates in the

intact organisms with a spatial resolution of F0.5mm. Both techniques require minimal or no samplepreparation, which ensures little artificial change inthe sulfur compound. Provided that the intensity ofthe exciting laser beam is kept sufficiently low, thesetechniques do not alter the speciation or crystallinityof the sulfur-bearing entity.

The laser Raman microprobe can detect spectro-scopically, with a spatial resolution of ;2 mm, thepresence of AsulfurB in the bacteria. Moreover, aRaman spectrum also documents the speciation of

Ž .the sulfur i.e., elemental or specific compound , itsŽstate of crystallinity coarsely crystalline, microcrys-

.talline, amorphousrglass , and—if crystalline—itsŽbasic crystal structure e.g., orthorhombic, mono-

.clinic . As demonstrated in the present study, laserRaman microprobe analysis is a powerful techniquefor the investigation of microbial metabolic reactionsand the pathways of element cycling in the crust andbiosphere, including diagenetic processes.

2. Application of Raman spectroscopy to micro-bial sulfur speciation

Individual Raman spectral bands represent theenergies of specific vibrational motions amongbonded atoms within a compound. As such, the

Žspectrum identifies the compound structurally e.g.,.calcite or aragonite , thereby permitting its composi-

Ž .tion e.g., CaCO to be inferred. Of importance here3

is the fact that elemental sulfur can take on multiplestructures, i.e., polymorphs, most of which are

Ž .metastable at room temperature Meyer, 1964 . TheRaman spectra differ for the individual crystal struc-

Ž .tures of elemental sulfur Edwards et al., 1997 , suchas orthorhombic, which is the stable form at roomtemperature, and monoclinic, which is metastable atroom temperature, but stable over a short tempera-

Ž .ture interval above about 96 8C Meyer, 1964 . TheRaman spectra also reflect differences in the config-uration among the atoms within a given molecularstructure. As shown in Fig. 2, the Raman spectrumof the common S ring configuration differs from8

that of the more rare S arrangement. Laser Raman6

microprobe spectroscopy, therefore, is a very desir-able technique to apply to the detection and charac-terization of sulfur vesicles in bacteria. It offers the

Ž .high spatial resolution on the order of micrometers

Fig. 2. Raman spectra of two different molecular configurations ofŽelemental sulfur, S and S . Spectrum of S is from unpublished8 6 6

.data, courtesy of Dr. Mark E. Jason, Solutia Company, 1997 .Raman spectra are plotted as intensity vs. a frequency term called

Ž y1 .wavenumber cm . It is not the absolute value of the frequencythat is plotted, but rather the Raman shift, i.e., the shift of thedetected frequency with respect to the frequency of the excitationlaser. Those recorded shifts reflect the frequency of vibration ofvarious motions of bonded atoms in a structure. A Raman spec-trum, therefore, is a molecular-structural fingerprint of a com-pound or element.


needed for pinpoint analysis, little sample prepara-tion, the ability to see exactly which feature is beinganalyzed, both chemical and structural information

Žand rapid results seconds to minutes, depending on.the level of structural-chemical detail required .

3. Experimental procedures

3.1. Sampling

All samples for these analyses were collected inMonterey Bay, CA, from the sediment–water inter-face in water depths between 900 and 1000 m. Thecollections were carried out with the Monterey BayAquarium Research Institute remotely operated vehi-

Ž .cle ROV Ventana supported by the research vesselPoint Lobos. Two cold seep sites were sampled for

Ž Xthe specimens analyzed: Clam Field 36844.0 N,X .12282.0 W , where clams, Thioploca spp. and other

bacterial mats were collected and Invert CliffŽ X X .36846.4 N, 12285.1 W , from which red and yellowBeggiatoa spp. were collected. Bacterial sampleswere collected through the use of corers 7 cm indiameter. Manipulator arms on the ROV insertedthese corers into the sediment on the order of 15 cmdeep, penetrating through bacterial mats on the sea

Ž .floor. Clams Calyptogena kilmeri also were col-lected with the manipulator arm. At the end of thedive, sediment cores and clams were placed togetherwith cold packs into thermally insulated containersfor transport to the laboratory. Upon return to shoreŽ .-2 h after completion of the dive , the bacterial

Ž .mats were separated in a cold-room at 10 8C fromthe sediment using cold, filtered seawater and gentlesieving. The mats were placed into 50-ml centrifuge

Žtubes, filled completely to exclude air as well as.possible with water that had overlain the sediment.

Clams also were maintained in cold, running seawa-Ž .ter for 2 days until prepared for shipping. Individ-

ual containers with bacterial mats and clams wereplaced into insulated packages together with coldpacks and shipped by overnight delivery service toWashington University, where they were placed im-mediately into a refrigerator at 2–4 8C.

Live organisms were stored in cold sea water untilthey were removed for microscopic analyses. Free-living bacteria were transferred directly to a micro-

scope slide, immersed in a few drops of sea water,and then covered with a microscope cover slip. Theclams were still alive when they were opened tosample the endosymbionts. The gills were dissectedand their contents smeared directly onto glass slides,after which they were covered with a few drops ofsea water and then protected with a cover slip. Thesamples were viewed and analyzed within minutes orhours of the slide preparation.

3.2. Analytical techniques

3.2.1. Optical microscopyThe samples on glass slides were viewed in trans-

mitted and reflected light with objectives of magnifi-cations up to 80= using an Olympus BH-2 polariz-ing microscope with a tungsten light source. The

Ž .relatively high refractive index 2.04 of elementalŽ .sulfur makes it strongly refractile high optical relief

and, thus, able to be imaged easily in transmittedlight. The high refractive index also accounts for therelatively high reflectivity of sulfur with respect toother transparent materials. The extremely large bire-

Ž .fringence of crystalline elemental sulfur 0.29 meansthat its birefringence can be detected under crossedpolarizers even in crystals that are only 0.5 mm thickŽ .Klein and Hurlbut, 1993 .

3.2.2. Laser scanning confocal microscopyŽ .Several individual trichomes filaments of Thio-

ploca were imaged using a Zeiss LSM 410 laserscanning confocal microscope equipped with objec-tives up to 60= and employing the 488-nm argonline for recording the epireflection image and the568-nm argon line for generating autofluorescenceimages of fluorescence emission wavelengths greater

Žthan 590 nm. The two types of images epireflection.and autofluorescence were recorded simultaneously

Ž .Fig. 3 . Each dual image was taken in one XY planeand multiple XY sections were taken throughout theheight of a trichome at 0.5-mm Z intervals. Theindividual XY images then were reassembled com-putationally to construct various three-dimensionalviews.

( )3.2.3. Laser Raman microprobe LRMTwo laser Raman microprobes were used for

analyses. Both instruments consist of a spectrometer


Fig. 3. Laser scanning confocal micrograph of a multicellular Thioploca trichome. Created by overlaying autofluorescence and epireflectionŽconfocal images. The fluorescence image, shown in red, is dominated by organic material of the thick cell walls and thin septa finely dotted

. Ž .lines . The epireflection image, shown in green, is dominated by the relatively reflective elemental sulfur; where the sulfur green is fullyŽ .enclosed in the cell wall red , the sulfur appears yellow in this image. Micrograph shows that most of the cell is a fluid-filled vacuole and

that sulfur vesicles are confined to cell walls and septa.

that is optically coupled to an Olympus research-grade microscope, which can be operated in trans-mission or reflection mode to view the sample and tofocus the laser beam onto or into the sample. Thelaser beam spot is about 1 mm in diameter at itstightest focus, but broadens several fold as it pene-trates the cover glass and enters the sample. Mostanalyses were done with a model S-3000 manufac-tured by Instruments SA. This is a multichannelRaman system with a triple spectrometer and em-ploying an intensified photodiode array detector. The

Žminimum recordable bandwidth i.e., full width at.half maximum with our typical instrumental config-

uration was 11–12 cmy1 in the spectral region ofinterest. Analyses were made using the 514.5 nmgreen line of an argon-ion laser for excitation. An80= -ultra-long working-distance objective was usedto deliver the laser beam to the sample. Spatialresolution was on the order of a few micrometers,and spectral resolution was approximately 7 cmy1.

The other Raman microprobe that was used is aHoloLab 5000, manufactured by Kaiser Optical Sys-tems. This is a spectrograph system employing atransmissive, volume holographic grating and a CCDdetector with 1024=256 pixels. A 632.8-nm HeNelaser and a 50= Olympus objective were used todeliver the laser to the sample. Spatial resolution wason the order of a few micrometers. In the region ofinterest, the spectral resolution was about 5 cmy1

and the minimum recordable bandwidth was 4–5cmy1.

The effect on the Raman spectra of the meltingand quenching of sulfur was determined by varyingthe power of the laser beam in the Raman micro-probe. For recording Raman spectra of sulfur invesicles of 1–2 mm in diameter, laser power at thesample had to be maintained at or below 1.5 mW.For both instruments, laser power in excess of 3 mW

Žat the sample led to immediate change peak broad-.ening in the Raman spectra and in the morphology


of the vesicle, indicative of melting–quenching.Spectra from both instruments were processed byGRAMSr32w software, commercially availablefrom Galactic Industries.

4. Results

4.1. Spatially locating sulfur bodies

Standard light microscopy, using both bright-fieldtransmission and epireflection, was used on all initialsample surveys. All the bacteria in this study areknown to produce intracellular sulfur vesicles orglobules. Bodies on the order of 1–2 mm diameterthat were round, colorless and of high optical reliefwere the targets of spectral analysis. In addition,laser scanning confocal microscopy was applied toseveral Thioploca samples primarily for the 3D lo-calization of the spherical, refractile bodies. Thereflectance images made of Thioploca predomi-nantly show the location of sulfur particles, whichreflect more light than biological tissue due to thehigh refractive index of sulfur. The red autofluo-

Ž .rescence excited by the 568-nm laser of membranematerial of the Thioploca reveals relatively thickouter cell walls and much thinner internal cell mem-branes, i.e., septa, within the trichomes. Photographs

Žthat are overlays of these two types of images Fig..3 show that most of the sulfur spherules are local-

ized in or adjacent to the cell walls and do not occurin the central portion of an individual cell, most ofwhich is a fluid-filled vacuole. In a few cases, sulfurspherules are located in the thin septum that isbetween cells and oriented perpendicular to the longaxis of the trichome. Thus, the confocal optical

Ž .micrographs Fig. 3 of the refractile bodies matchŽ Ž ..the TEM images Fig. 1 B that show the location

of the electron-dense spheres andror apparent drop-out regions.

ŽBoth optical microscopy standard and laser scan-.ning confocal and laser Raman spectroscopy were

used in a survey mode to visually pinpoint andspectroscopically confirm the identity of sulfur vesi-cles in four types of sulfur-precipitating bacteria.Fig. 4 contains photographs of examples of themicrometer-scale refractile spheres that yield the Ra-

man spectrum of elemental sulfur. Several types ofmorphologies of putative sulfur vesicles were inves-tigated. In trichomes of Thioploca and Beggiatoa, as

Ž Ž .expected, the round 1–2 mm spherules Figs. 4 a ,Ž . Ž ..b , 5 a are the sulfur bodies. Standard opticalmicroscopy of such spherules under crossed polariz-ers showed no birefringence, thereby providing evi-dence that each spherule was not a single crystal ofsulfur. However, in similar Thioploca and Beggiatoasamples that had remained on the microscope slides

Ž .for relatively long times from 1 to 48 h , the sulfurspherules clearly underwent physical changes. First,the spherules aggregated into dark clusters on the

Ž Ž ..order of 7–10-mm diameter Fig. 5 b . Later, theclusters developed into individual, clear, polygonal

Ž Ž ..crystals Fig. 5 c , whose response under crossedpolarizers showed them to be birefringent, singlecrystals. Raman spectroscopy confirmed that thecrystals, as well as the spherules, were elemental

Ž .sulfur see below .Other bacterial mats contained apparently identi-

cal colorless filaments in close association. In mostcases, the filaments appeared empty of inclusionsŽ .vesicles ; however, a few were packed with what

Ž Ž ..appeared to be submicrometer granules Fig. 4 e .In transmitted light under crossed polarizers, no bire-fringence was detected in the granules. However,they clearly were more reflective than the surround-ing tissue when viewed in reflected light using a

Ž Ž ..standard microscope Fig. 4 f . These granules alsogave the Raman spectroscopic signature of elementalsulfur.

The clam gill from C. kilmeri provided the mostchallenging sulfur vesicles to locate. Transmittedlight revealed several sizes of colorless, vesicle-like

Ž Ž . Ž ..bodies in the gill tissue Fig. 4 c , d , ranging fromŽ-1 to 10s of mm in diameter cf. TEM view in Fig.

Ž . Ž ..1 C , D . The most notable optical features, how-ever, were rounded, black bodies of 30–40 mm in

Ž Ž ..diameter Fig. 4 c . In some sample smears onslides, these bodies blackened more than half of afield of view that was hundreds of micrometers wide.In a few cases, it was apparent that the smallest of

Ž .the colorless vesicles F1 mm underwent variousstages of aggregation into thin swaths and ultimately

Ž Ž ..into the large, black spheres Fig. 4 d . The opacityof the sphere, as viewed in bright-field transmission,presumably is due to the high amount of light scat-


Ž .Fig. 4. Standard photomicrographs of four types of sulfur-oxidizing bacteria. All photographs taken in transmitted light, except for Fig. 5 f ,Ž . Ž .which was taken in reflected light. a Thioploca trichome; minute sulfur vesicles appear as spherical bodies with dark outlines high relief .

Ž . Ž . Ž .b Beggiatoa trichomes with sulfur vesicles. c and d Endosymbionts in gill of vesicomyid clam C. kilmeri. d Illustrates various stages ofaggregation of individual, 1-mm, colorless, spherical bacteria into large, opaque, subspherical masses like the two black bacteriocytes in Fig.Ž . Ž . Ž . Ž .4 c ; compare to TEM images in Fig. 1 C and D . e and f Thin filamentous bacteria in which the approximately horizontal filament

Ž . Ž .appears empty, whereas the approximately vertical filament is filled with submicrometer-sized granules of sulfur. e In transmitted light. fThe same field of view in reflected light, demonstrating the relatively high reflectivity of elemental sulfur.

tering from the densely packed individual minutevesicles. Raman spectroscopy showed a very strongsignal for sulfur in the black spheres, whereas at-tempts to get a signal from individual submicrometervesicles were unsuccessful. A string of three suchvesicles, however, did give the Raman spectrum ofelemental sulfur. It, thus, appears that the 1-mmvesicles are the individual sulfur-precipitating bacte-ria and that the large black bodies are bacteriocytesinto which the bacteria, containing vesicles, have

Ž Ž .aggregated Fig. 1 D , this paper; cf. Vetter, 1985;.Fiala-Medioni and Le Pennec, 1988 . This interpreta-´

tion is supported by microscopic observations inreflected light, in which both the individual sulfurvesicles and the large black bacteriocytes show theexpected high reflectivity of elemental sulfur.

Every effort was made to assure that the sulfur-precipitating organisms were alive directly beforeRaman analysis proceeded in order to reduce the

Ž .possibility of recrystallization of the sulfur.


4.2. More detailed characterization of sulfurspherules in Thioploca and Beggiatoa

Due to the long-standing discussions about thephysical and chemical structure of the 1–2 mm

Žspherules in Thioploca and Beggiatoa Shively et.al., 1988; Steudel, 1989; Javor et al., 1990 and to

the fact that these vesicles were the largest in oursample suite, we pursued a more detailed Ramanspectroscopic characterization of them. Our interpre-tations are based on approximately 120 Raman anal-yses of spherulesrvesicles in multiple trichomes fromspecimens collected on seven different occasions.Only the elemental form of sulfur was detected inthe Raman spectra of these spherules; however, thelaser power on the sample was extremely low, asindicated above. There was no evidence of othersulfur compounds in the spherules, such as sulfides,sulfonates and sulfates, which are also strong Raman

Ž .scatterers Lin-Vien et al., 1991 . In molecular–structural terms, the elemental sulfur is arranged in

Žthe typical eightfold sulfur ring configuration Fig. 2,.upper . Each spherule is very finely crystalline, as

distinguished from either coarsely crystalline oramorphousrglassy. These spectral results are thesame for spherules in both the Thioploca and Beg-

Fig. 5. Standard photomicrographs in transmitted light showingŽthe aging and deterioration process in sulfur vesicles dark-out-

.lined, round bodies from filamentous sulfide-oxidizing bacteria.Ž .a Fresh trichome of white Beggiatoa; vesicles quite evenlydistributed; some vesicles appear spatially isolated. Raman spec-

Ž .trum compare to Fig. 6, middle indicates extremely fine-grained,Ž .microcrystalline, solid, elemental sulfur. b Trichome of yellow

Beggiatoa in seawater after sample has been unrefrigerated andexposed to air for more than 1 h. Note how sulfur vesicles have

Ž .aggregated and become more elongated, less spherical. c Adifferent trichome of yellow Beggiatoa after 48 h on a glass slidewithout refrigeration. Optically isotropic sulfur spherules almostcompletely replaced by birefringent polygons 5–10 mm long ofdominantly two shapes: elongated, needle-like and rhombohedralto rounded. Both give Raman spectra of elemental, crystalline

Ž . Žsulfur cf. Fig. 6, bottom . Under crossed polarizers not shown.here , rhombohedral crystals show 18 white interference color. In

contrast, needles show 18 yellow interference color; they areŽ .optically length-fast length-negative and show oblique extinc-

tion. Interestingly, the latter suggests that the needles are mono-Žclinic rather than orthorhombic the stable structural form of

.sulfur at room temperature and below .


giatoa samples that we studied. Our interpretationsare based on comparisons of Raman spectra of wellcharacterized sulfur samples with those of spectrataken on spherules within individual trichomes, asdescribed in detail below.

The middle spectrum in Fig. 6 is typical of thosetaken on fresh Thioploca and Beggiatoa. Compari-son of this spectrum with those in Fig. 2 confirmsthe spherule material to be elemental sulfur in the S8

configuration. Comparison of the middle and bottomspectra in Fig. 6 highlights the need for care in the

Fig. 6. Raman spectra of sulfur in Thioploca trichomes, analyzedunder different instrumental and biological conditions. Middlespectrum represents ideal conditions for Raman spectroscopicanalysis, in which the trichome is fresh, and minimal laser powerŽ .1.5 mW is focused into the sulfur vesicle for only a few secondsŽ .cf. Fig. 7, middle . Upper spectrum shows how a 10-fold increasein laser power causes the sulfur vesicle to melt and undergo

Ž .structural disruption cf. Fig. 7, upper . Bottom spectrum wasŽ .taken on recrystallized sulfur cf. Fig. 7, bottom in a trichome

that had been stored in a tube partially filled with glutaraldehydeŽ Ž ..and with air in the head space cf. Fig. 5 c .

choice of material to analyze. Even in trichomeswhose biological tissue appears well preserved byformalin or glutaraldehyde, the physical–chemicalnature of some of the spherules may have beenchanged, in some cases converted to single crystals

Ž .by dissolution and recrystallization Fig. 5 . Aggre-gation of the spherules and, especially, the presence

Ž .of polygonal typically rhomb-like or needle-like ,Ž Ž ..colorless, birefringent bodies as in Fig. 5 c , indi-

cate transformation of the original material into sin-gle crystals. Comparison of the bottom spectrum inFig. 6, taken on a 7-mm sulfur crystal in a preserved

Žtrichome, with that of the 2-mm fresh spherule mid-.dle spectrum indicates that these spectra can provide

information on the degree of crystallinity of thesulfur.

There are two major ways in which the spectra ofsulfur samples of various degrees of crystallinitydiffer, i.e., have different length scales of continuityin the ordered arrangement of S rings. Firstly,8

although the Raman peak positions detected in acrystal of material will not change as the crystal isrotated, in most cases, rotation will produce a changein the relatiÕe intensities of the peaks. This is due tothe anisotropy in the polarization in the Ramanspectrum of crystalline sulfur, which arises from thesymmetry properties of its particular crystallinestructure. Our work confirms this effect in coarsecrystals of sulfur. We observed, however, no majordifferences in the peak–height ratios in spectra takenon individual bacterial spherules, thereby eliminatingthe possibility that each was a single crystal of sulfuror a bundle of parallel crystals. The relative con-stancy of peak–height ratios is compatible with ei-ther a random arrangement of microcrystals or aglassy state. Another crystallinity-sensitive spectralfeature, namely, the Raman band width, was used todistinguish between the latter two possibilities.

Fig. 7 demonstrates the Raman spectral changesŽobservable see especially the spectral region ofy1 .400–600 cm in samples of pure elemental sulfur

of various degrees of crystallinity. The bottom spec-trum was taken on a natural geological sample ofcentimeter-scale sulfur crystals from Cianciani,Sicily. Some of this same material was heated untilmolten, cooled at different rates, evaluated for itscrystallinity under a standard microscope usingtransmitted and reflected light and then analyzed


Fig. 7. Raman spectra of elemental sulfur, artificially processed toproduce three different degrees of crystallinity. Upper spectrumtaken on sulfur that had been heated to a liquid and then quenchedto a glass. Middle spectrum taken on sulfur that had been heatedto a liquid and then cooled rapidly to form quench crystals on theorder of 10s of mm in length, as observed in reflected light.Bottom spectrum taken on natural sulfur crystal, about 1 cm long,

Ž .from Cianciani Sicily . Differences in degree of crystallinity areespecially apparent in 400–600 cmy1 spectral region. Note bandbroadening and decreased resolution of peaks in the progression

Ž .from most crystalline to least crystalline amorphous sulfur.Compare these spectra with those in Fig. 6.

Žwithin a few hours to avoid the possibility of recrys-.tallization . The most quickly cooled liquid produced

an isotropic glass whose Raman spectrum is at thetop of Fig. 7. The liquid that was cooled moreslowly, whose spectrum is in the middle of thefigure, produced a mass of quench crystals on theorder of 10s of mm in length. In the 400–600 cmy1

Žspectral region, the increase in the band width mea-.sured as full width at half-maximum intensity from

coarsely crystalline to microcrystalline to glassy sul-fur is readily seen. Comparison of these three spectra

with the middle spectrum in Fig. 6 indicates that thesulfur in fresh spherules is very finely crystallinerather than amorphousrglassy. Without detailed cali-bration of Raman spectral bandwidth with respect to

Ž .crystallite size as determined by TEM , one cannotcalculate the actual size of the crystallites in thespherules. It is apparent by comparison of the middlespectra in Figs. 6 and 7, however, that the Thioplocasulfur has a smaller crystallite size than the artifi-cially produced quench crystals.

The importance of both the biological and miner-alogical stability of the sample also is demonstratedby the spectra. Comparison of the middle and lowerspectra in Fig. 6 and comparison of both of thosespectra with the spectral calibration suite in Fig. 7demonstrate that sulfur in the altered, polygonal

Ž .bodies bottom spectrum found in aged samplesstored in seawater or glutaraldehyde is recognizablymore crystalline than in the fresh spherules. The carenecessary in analyzing individual spherules also isreflected in the top spectrum of Fig. 6, whichdemonstrates the band broadening that occurs whentoo intense of a laser beam is focused onto a smallspherule and causes the latter to melt. We, therefore,kept the laser power to less than about 1.5 mW at thesample surface in order to perform detailed, nonde-structive structural analyses. Because of the possibil-ity that even a 1.5-mW laser beam could heat these

Žminute samples to over 90 8C approximate transi-tion temperature for orthorhombic–monoclinic tran-

.sition , we cannot say with certainty which crystalstructure of S is present.8

5. Discussion

5.1. Sulfur speciation, crystallinity and solubility

As detailed above, our Raman spectral data docu-ment the sulfur to be in a microcrystalline state,which is not in agreement with earlier XRD-basedinterpretations of vesicles from Chromatium bacte-

Ž .ria, which Hageage et al. 1970 inferred to be totallyamorphous or even liquid-like. Their statement hasbeen reiterated numerous times over the years andapplied to a wide variety of sulfur-precipitating bac-teria, apparently without additional XRD investiga-


Žtion e.g., Lawry et al., 1981; Vetter, 1985; Shivelyet al., 1988; Steudel, 1989; Javor et al., 1990; Prange

. Ž .et al., 1999 . One possible biological explanationfor the difference in our findings concerning thephysical state of the sulfur vesicles is that differentgenera of bacteria may handle sulfide oxidation dif-

Žferently consider the filamentous sulfur precipitatedby Arcobacter species; Taylor and Wirsen, 1997;

.Taylor et al., 1999 . There is also a possible analyti-cal explanation for the difference between the Ra-man-based interpretation of the vesicles as micro-crystalline and the XRD-based interpretation of aliquid-like or amorphous state. The discrepancy mayreflect the difference in sensitivity of Raman spec-troscopy and X-ray diffractometry to the crystallinityof materials over specific length scales of latticecontinuity. X-ray diffraction arises from the coher-

Ž .ence of the lattice planes within a crystalline mate-rial, whereas Raman spectroscopy interrogates themore local molecular environment over a maximumof a few unit cells of the crystal. Our previous

Žresearch has demonstrated that even zircons whoselattice can be damaged by radioactive decay of con-

.tained uranium that have been labeled as AX-rayamorphousB yield Raman spectra indicative of crys-

Žtalline, nonglassy material Wopenka et al., 1996; B..Wopenka, personal communication, 2000 .

Our results also differ from those of Prange et al.Ž . Ž .1999 , who analyzed by XANES the bacteria di-rectly rather than removing the sulfur spherules andthen analyzing them. They conclude from compari-son of XANES spectra of several phototrophic bacte-ria with those of known sulfur standards that thespherules probably are composed of long chains ofsulfur that are terminated by C-bearing groups. Theyspecifically rule out the presence of cyclic forms ofelemental sulfur and of polythionates. The lack ofcompatibility between their results and those of thepresent study may reflect the difference in the bacte-rial group under analysis or the difference in samplehandling before analysis.

Among the interpretations of the vesicle contents,Žbased on various indirect analyses such as rapid rate

of vesicle metabolism and fine-scale morphology of.extracted vesicles , is the notion that the vesicles

Ž .contain a AwettedB Hageage et al., 1970 , Ahy-Ž .dratedB Guerrero et al., 1984 , or otherwise hy-

drophilic sulfur species that Ais not a form that is

Žtypical of orthorhombic sulfurB Shively et al., 1988,.p. 83 . Our interpretation of our Raman spectra

provides an alternative model that is based on directmeasurements within the bacteria. Spectral evidenceindicates that each small sulfur vesicle is comprised

Žof yet smaller crystallites of indeterminate actualsize in the absence of Raman spectra correlatedagainst standards with known length scales of crys-

.talline order . We believe that the microcrystallinityof the elemental sulfur in the vesicles accounts for itsobserved high reactivity, because a high surface–volume ratio promotes rapid dissolution of a mate-rial.

5.2. Problems with optical artifacts in analyzingsulfur in spherules Õia microscopy

Our optical microscopic observations also differŽ .from those that Hageage et al. 1970 made on

vesicles in Chromatium and from those of VetterŽ .1985 on symbionts in the gills of L. annulata. Inviewing sulfur vesicles within fresh samples of Thio-ploca and Beggiatoa under crossed polarizers intransmitted light, we did not detect the Maltese-crosspattern that was photographically documented by

Ž .Hageage et al. 1970 , which they realized was sug-gestive of the radial alignment of small crystals.

Ž .Apparently, Hageage et al. 1970 perceived a con-flict in interpretation between an amorphous state, asindicated by the XRD data, and a crystalline state, asindicated by the optical microscopy data. It may bethis perceived conflict that led them to make state-ments that have persuaded subsequent researchers toclaim that the spherules contain elemental sulfur in a

ŽAliquid crystalline stateB Vetter, 1985, p. 36; Javoret al., 1990, p. 236; similar statement in Lawry et al.,

.1981 .We did, however, observe a Maltese-cross pattern

in the sulfur spherules under crossed polarizers inreflected light, using the vertical illuminator of the

Ž .microscope. Any particularly, small spherical ob-ject that has moderate reflectivity, regardless of

Žwhether the sphere contains amorphous optically. Ž .isotropic or crystalline probably birefringent mate-

rial, will produce a Maltese cross under crossedpolarizers in reflected light simply due to the polar-ization rotation that occurs when light reflects off a


sphere. In small spherules, therefore, the observationof a Maltese cross under crossed polarizers in re-flected light offers no insight into the material’sŽ .lack of crystalline structure. A weaker Maltesecross also may be visible in spherules under crossedpolarizers in transmitted light by use of very intenseillumination in conjunction with a cover slip over the

Ž .spherules J.J. Freeman, unpublished observations .In this case, the difference in refractive index be-tween the sample and its overlying cover slip causesdownward reflection of some of the transmitted lightback onto the spherules, thereby imitating the opticalgeometry of reflected-light microscopy. These obser-vations are further confirmation that the high refrac-tive index of sulfur and the extremely small size ofthe sulfur vesicles make it difficult to characterizethe vesicle contents by optical microscopy. It ispossible that the physical–chemical nature of sulfurin vesicles differs among different bacterial groupsŽ .cf. Strohl et al., 1981; Shively et al., 1988 . How-ever, optical microscopic interrogation of the vesi-cles is not a reliable way of exploring those differ-ences and similarities.

The documented rapidity of recrystallization thatŽoccurs in sulfur spherules Hageage et al., 1970;

.Vetter, 1985; this work warns against interpretationof the composition and internal morphology of vesi-cle material based on air-dried samples or evenfixed samples that were removed from their host

Žmany days prior to analysis Lawry et al., 1981;.Fiala-Medioni and Le Pennec, 1988 . Direct analyses´

or techniques developed specifically to preserve theŽoriginal sulfur product as distinguished from pre-

.serving the biological tissue are required to charac-terize vesicles in various sulfur-precipitating bacte-ria.

5.3. Biological–biochemical importance of the physi-cal–chemical state of sulfur in spherules

ŽKnowledge of the chemical speciation in this.case, elemental S8 of the sulfur product helps to

constrain the specific reaction that caused it to pre-cipitate. Compound specification also allows for theevaluation of the stability, including solubility, ofthat form of sulfur in the biological and seawaterenvironments. In turn, recognition of the operable

Ž .reaction s permits inference about the limiting com-pounds to growth and the chemical species thatcould be monitored as tracers of Thioploca and

Ž .Beggiatoa metabolism cf. Otte et al., 1999 . Wephotomicrographically monitored Beggiatoa over aperiod of days after they had been removed fromtheir seafloor environment, but maintained in 50-mlclosed containers of seawater at 2–4 8C. Monitoring

Žof several specific filaments under unknown condi-.tions of sulfur fugacity over time showed that the

size of the vesicles decreased and that some of theterminal cells of the filaments ultimately becameempty of sulfur vesicles. Our observations suggestthat sulfur vesicles in Beggiatoa dissolve and pre-sumably are utilized by the bacteria in the absence ofthe H S that typically sustains them on the seafloor2Ž .Vetter, 1985 . Putative sulfur vesicles in Beggiatoaalba, expand and collapse in the presence and ab-sence, respectively, of exogenous sulfide, suggesting

Žmetabolite consumption of sulfur stores Lawry et.al., 1981; Shively et al., 1988 . We conclude from

our observations and those of others that high solu-bility and chemical reactivity of stored sulfur areimportant to the bacteria, and that these propertiesare imparted by the microcrystalline state of thesulfur.

5.4. Mineralogical–geochemical importance of thephysical–chemical state of sulfur in spherules

The fact that compound-specification of the sul-fur-bearing spherules helps define the precipitationreactions means that one can evaluate how much freeenergy of reaction is available for use by the sulfur-

Žoxidizing bacteria Somero et al., 1989; Nelson andHagen, 1995; McCollom and Shock, 1997; Kargel et

.al., 1999 . It is important geologically that thesebacteria can provide an efficient concentrating pro-cess for sulfur in the sediments. Our Raman spectro-scopic and optical microscopic observations suggestthat, in the absence of bioabsorption, the microcrys-talline sulfur in the vesicles is highly susceptible torecrystallization and bacterial disproportionation to

Ž .reactive sulfide qsulfate , which can lead to reac-tion with available iron in the seafloor sediments to

Žproduce iron sulfides Javor et al., 1990; Thamdrup.et al., 1994; Ferdelman et al., 1997 . These bacterial


processes may explain, in part, why reduced marinesediments are such an important source of diageneticiron sulfide minerals, which, in turn, develop into

Ž .pyrite-rich shales e.g, Berner, 1970, 1984 .

6. Conclusions

Laser scanning confocal microscopy and laserRaman microprobe spectroscopy both can be used tospatially locate sulfur in vesicles as small as 1-mmdiameter in intact biological samples. In more de-tailed studies, Raman spectroscopy can be used fur-

Žther to characterize the sulfur chemically identify.species of the compound or element and struc-

Žturallyrphysically crystal structure and degree of.crystallinity . Fortunately, sulfur is an excellent Ra-

man scatterer, which allows small volumes of it to bedetected despite the fact that laser irradiation powermust be kept low in order to prevent the sulfur frommelting. Operating in the location-identificationmode, we identified the contents of vesicles in all

Žfour types of our samples Thioploca, Beggiatoa,very narrow bacterial filaments and symbiont in C.

.kilmeri as elemental sulfur. Our studies suggest thatthe spectral imaging capabilities of the newest Ra-

Ž .man microprobes not used in this study wouldpermit the distribution of elemental sulfur to bemapped within these organisms with a spatial resolu-tion of a few micrometers.

More detailed Raman spectral analyses furtherdemonstrate that the 1–3 mm vesicles in both Thio-ploca and Beggiatoa are dominated by microcrys-talline, elemental sulfur in the stable S ring configu-8

ration. The exact size and specific arrangement ofthe minute sulfur crystallites within the vesicles isnot known. The fact that each individual vesicleis not a single crystal of sulfur or even a group ofaligned microcrystallites in parallel or radialŽ .spherulitic array is confirmed by the lack of ob-servable birefringence when the spherules are viewedunder crossed polarizers. Although other organic andinorganic sulfur compounds also are known to bestrong Raman scatterers, no species other than ele-mental sulfur was detected. This finding confirmsthat the vesicles are not dominated by a homoge-neous organo-sulfur compound, which was one pos-

Ž .sible model explored by Steudel 1989 . However,

our data are compatible with another model of SteudelŽ .1989 in which microcrystallites of elemental sulfurdominate the vesicles, but are stabilized by a thinorganic membrane.

There are multiple ramifications to the high solu-bility and reactivity that are imparted to the vesiclesulfur from its minute crystallite size. Biochemically,the sulfur can be utilized readily by the bacteria.Mineralogically, sulfur associated with the bacteriacan enter the sediment and water columns after theirdeath and be reduced to sulfide or react directly withdissolved iron to produce diagenetic iron sulfideminerals. The high reactivity of the sulfur also is achallenge in its characterization, because analysesmust be made before the original material reacts tochange either its speciation or its degree of crys-tallinity.

Laser Raman microprobe spectroscopy offers newways to study very small volumes of both liquid andsolid metabolic products directly within bacteria,thereby reducing concerns about artifacts, which canbe introduced by some biochemical techniques. Manyquestions remain about the details of sulfur-basedchemoautotrophy, particularly, concerning the meta-bolism of symbionts, which cannot be cultured. Ap-plication of Raman spectroscopy to these bacteria,

Ž .both through pinpoint analyses as discussed hereand spectral mapping, may elucidate the specificchemical pathways and energetic gains of individualmicrobes by measuring the presence andror concen-tration of identifiable sulfur intermediate and end-products.

Acknowledgements

We would like to thank Drs. Alian Wang andBrigitte Wopenka for their help with some of theRaman analyses, as well as Dr. Wang and Dr. LarryHaskin for the use of their Raman instrument. Wealso thank the crew of the RV Point Lobos and thepilots of the ROV Ventana for their assistance incollecting the samples. We greatly appreciate thehelp of Dr. Peter Brewer in stimulating the discus-sions that led to this project and the efforts of Drs.Johnson Haas and Jeremy Fein in organizing andediting this special volume. Thoughtful reviews by


Drs. Michael Bottcher, Jeffrey Kargel and an anony-¨mous reviewer were very helpful. A portion of thiswork was funded by a grant from the Packard Foun-dation to Dr. J.P. Barry.

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