Crystalline sulphur deposits on the tube of the hydrothermal vent worm Alvinella pompejana

Camp. Biochn.

Printed in Great

Physiol. Vol. 102A, No. I, pp. 71-77, 1992 Britain

0300-9629/92 55.00 + 0.00 0 1992 Pergamon Press plc

CRYSTALLINE SULPHUR DEPOSITS ON THE TUBE OF THE HYDROTHERMAL VENT WORM

ALVINELLA

POMPEJANA

STEPHENHUNT Department of Biological Sciences, University of Lancaster, Bail&, Lancaster, LA1 4YQ, U.K. Telephone: (0524) 65201; Fax: (0524) 638-06 (Received 22 Auga.rt 1991) Abstract-l. Microscopy of the inside wall of the dwelling tube of the polychaete worm Alvinella pompejana reveals the presence of crystals in association with filamentous bacteria. 2. Microprobe analysis shows the crystals to be sulphur with no other element above mass number 23 accompanying this in appreciable amount. 3. Crystalline, rhombic, sulphur is not found on the tube’s outer face although granules rich in sulphur and in what is probably iron(H) and zinc sulphides are present. 4. The sulphur is presumed to be produced by sulphide-oxidising bacteria. 5. Crystallization may occur as liquid crystal, cytoplasmic inclusions are externalized.

INTRODUCTION

in distilled water, allowed to air dry, mounted with graphite paste cement onto carbon support stubs and then either sputter-coated with gold or coated with carbon evaporated from an arc in a rotary coating unit for X-ray microprobe analysis. Microscopy was performed with a Jeol JSMSOA scanning electron microscope at an operating voltage of 15 kV. Elemental analyses were made on the same instrument at an excitation voltage of 15 kV and with a Kevex energy dispersive analysis system having a gain setting of 20 eV per channel and a preset live time of 200 sec.

In this paper the detection of elemental sulphur in crystal form and apparently of biological origin, deposited upon the inside wall of the dwelling tube of a polychaete worm, Alvinella pompejana, which lives at abyssal depths, in the Pacific Ocean, in close proximity to the recently discovered sub-marine hydrothermal vents of the Galapagos Ridge is described, The sulphur crystals occur in association

with mats of filamentous bacteria which are probably chemolithotrophs utilising exogenous hydrothermal hydrogen sulphide as an energy source. The production of well-defined crystals of sulphur in an aqueous medium is unusual and, since the hydrophobic character of the element must present particular problems for an organism oxidatively metabolising hydrogen sulphide to sulphur within its cytoplasm, this observation is of some interest from the aspect of an organism’s capacity to handle the element in bulk as well as in illuminating one aspect of these Thiojix-type filamentous bacteria’s sulphur metabolism, which is at present only partially elucidated. This observation may suggest that these chemolithotrophs use hydrogen sulphide as a primary energy source and may store elemental sulphur extracellularly as a second energy source for oxidation during times of low hydrogen sulphide availability. Their capacity to do this is of importance to the host animal whose own metabolic economy is probably very considerably dependent upon these bacteria. MATERIALS

RESULTS

Figures 1 and 2 are representative scanning electron micrographs of the external and internal surfaces, respectively of the tube of Alvinella. The chemical composition and ultrastructure of the largely proteinaceous but partially mineralized tube of Alvinella has been described previously by Gail1 and Hunt (1986, 1991). The outer surface is coarsely and irregularly textured with numerous large and small desposits scattered unevenly over it. Some bacterial filaments are present but not in any great number. Some of the larger deposits are smoothly nodular but most have no regular form. Figure 3A shows the energy dispersive X-ray profile obtained by probing one of the large round grains seen in Fig. 1 with theelectron beam. The profile indicates that this is composed largely of sulphur and iron suggesting that it is an iron sulphide deposit. This is probably iron(I1) sulphide as acidification of the tube releases hydrogen sulphide (Gail1 and Hunt, 1986). For comparison, Fig. 3B shows the X-ray profile obtained from a relatively smooth area between the granular deposits. Here sodium, phosphorus, sulphur, chlorine and iron with some potasium and calcium are the main elements. The other small granular deposits on the outer surface either have elemental compositions similar to the larger rounded deposits, or to the general surface or else show an elemental profile similar to that shown in Fig. 3C. This latter, from the probing of an irregularly

AND METHODS

Tubes of Alvinella pompejana were a gift from Dr Jean Vovelle of the Laboratory of Marine Invertebrate Histology and Cytology at the Universite Pierre et Marie Curie, Paris, and were collected during the French BIOCYATHERM expedition at a depth of 2600m near to 13”N, 104”W in April 1982. They had been preserved in formol-saline. Scanning electron microscopy was carried out on pieces of tube wall about 4 mm square which had first been rinsed CBPA IOZ,l-i=

71

72

STEPHEN HUNT

Fig. 1. Scanning electron micrograph of the outer face of the dwelling tube of the hydrothermal vent polychaete worm Alvinella pompejana. The short arrow indicates one of the larger iron/sulphur-rich deposits for which the microprobe analysis of Fig. 3A is a typical example. The long arrow indicates an unobstructed area of the basic tube material for which the analysis of Fig. 3B is typical and the broad arrow head refers to a sulphur-rich deposit for which the analysis of Fig. 3C is typical.

angular particle shown in Fig. 1, has a very high sulphur content in relation to the other elements present and many therefore be a small impure crystallite of elemental sulphur. A semi-quantitative tabulation of individual elemental contributions based upon the count rate data from Figs 3A-C is given in Table 1.

The appearance of the inner face of the tube (Fig. 2) is very different. A feltwork of bacterial filaments densely covers most of the surface although bare patches can be found. Here, the elemental composition is rather similar to that of the unobstructed basal material of the outer face (Fig. 4A and Table 1)

Fig. 2. Scanning electron micrograph of the inner face of the Alvinella tube. The short arrow indicates an area of tube material unobstructed by either bacterial filaments or appreciable granular deposits for which the analysis of Fig. 4A is a typical example. The long arrow indicates a large sulphur crystal for which the analysis of Fig. 4B is typical.

13

Sulphur in hydrothermal vent worms

4A

L

Fe

Zn

3B

4B

Fig. 4. Microprobe

analysis of the internal face of the

Aluinellu tube. (A) An area essentially free of granular deposits and bacteria. (B) One of the large crystals.

Fig. 3. Microprobe analyses of three different characteristic features of the outer wall of the Aluinella tube. (A) One of the large rounded granules. (B) An area of the tube material essentially free of granular deposits. (C) One of the small irregular granules. The analysis is energy dispersive and the abscissa1 axis is in terms of X-ray energy and is a measure of increasing atomic number while the ordinate axis is a measure of received X-ray photon counts at the detector. Table

although there is less chlorine relative to phosphorus and sulphur and a trace of zinc which, like the iron, is probably present as the sulphide (Gail1 and Hunt, 1986). Small spherical granules and clumps of amorphous material entrapped among the filaments correspond in elemental composition to those found on the outside surface. But the most prominent feature of the inside face of these specimens of tube are the large crystals which force their way up through the mat of bacterial filaments so that they appear only to be tethered by thin ropes of material. Microprobing these crystals reveals that they are apparently composed of almost pure sulphur although of course the microprobe does not reveal

I. Elemental composition of the outer and inner faces of the dwelling tube of the polychaete annelid worm

determined by X-ray microprobe analysis. Values are as emitted X-ray counts for each integrated elemental peak as a percentage of total counts integrated across the whole energy spectrum with background subtracted. For conditions see text. These values relate to Figs 2 and 4 A/vine/la pompejano

Outer Surface Particle-free Large round granule* areat Na Mg Br P s Cl K Ca Fe Zn

0.41 0.34 0.1 I 0.47 78.24 0.00 0.16 0.03 20.14 0.09

4.99 1.10 0.00 23.13 17.75 41.61 1.72 2.07 7.62 0.01

Small irregular grain1 0.84 0.20 0.07 1.72 93.59

1.08 0.27 0.56 1.61 0.07

Inner Surface Particle-free areas 3.25 3.02 0.63 27.55 22.59 28.73 2.02 2.59 9.40 0.29

Crystal

I/

0.07 0.00 0.06 0.00 99.68 0.00 0.10 0.00 0.10 0.00

The analyses are of structures on the tube, indicated on Figs I and 2 as follows and designated accordingly by the indicated superscripts in the Table headings. *Fig. I Short arrow. tFig. I Long arrow.$ Fig. I Broad arrow head.8 Fig. 2 Short arrow.1) Fig. 2 Long arrow. From left to right the columns in the Table refer, respectively to Figs 3A, B, C, 4A and B.

74

STEPHEN

elements lighter than sodium (Fig. 4B and Table 1). Treatment of pieces of tube with carbon disulphide extracts pure sulphur in solution and removes any evidence of these large crystals (Gail1 and Hunt, 1986). It seems certain therefore that the crystals are indeed of elemental sulphur in free form and that from their morphology they are in the form of the more stable rhombic allotrope. DLSCUSSION

Crystals of elemental sulphur are therefore found on the inside face of the dwelling tube of Alvinella and not on the exterior. This points to a specific activity within the tube resulting in their production. The most likely explanation of this lies with the exosymbiotic, epibiotic, bacterial flora which is associated with the worm’s integument and with the tube wall (Gail1 and Hunt, 1991). The inhalent respiratory stream drawn through the tube by the worm’s ciliated gills benefits the bacterial population and this in its turn presumably provides the worm with a source of nutrients. These bacteria are of a type utilizing inorganic sulphur. The discovery in the early 1980’s of a prolific fauna at ocean depths of 200&3000 m and close to high temperature venting regions of subducted water caused considerable surprise (Laubier and Desbruyhes, 1984, 1985; Brock, 1985). These animals survive not only low oxygen tension but more remarkably the total lack of access to solar energy or photosynthesis. An important nutrient source for many of the sedentary animals lower down the food chain arises through the activities of chemolithoautotrophic bacteria which may be either free-living or in symbiotic relationship with them (Felbeck and Somero, 1982; Jannasch and Wirsen, 1985). In these bacteria a primary source of energy, for complex carbon compound synthesis, has proved to be the hydrogen sulphide which can often be present at high concentration in the vent waters. Oxidation of hydrogen sulphide to sulphur-and thence to sulphate in some cases-provides a means to anaerobic oxidative metabolic provision of energy for carbon dioxide fixation through the Calvin cycle. The hydrogen sulphide has its origin in subducted sulphate being reduced by ferrous iron during its contact with the hot magma (Rona, 1986). The economy of the hydrothermal vent communities pivots upon the autotrophic bacterial populations therefore, as well as upon the continued flow of mineral nutrients and oxidisable simple carbon molecules in the vent water flows. It is possible to group the sulphur-oxidising bacteria of the hydrothermal vent communities into three major types and these have been categorized by Tuttle (1985) as acid-producing obligate chemoautotrophs, facultative auto or heterotrophs and sulphur bacteria which are often large and filamentous. The first two groups are small, Gram-negative, rod-shaped and often free-living organisms which in the case of the first group variously oxidise sulphide, elemental sulphur, thiosulphate, polythionates and sulphite to sulphate. This group are genuine primary producers as they utilize the energy derived from these oxidations to convert carbon dioxide into or-

HUNT ganic molecules using the Calvin cycle. The second group oxidise sulphide or thiosulphate to polythionates and many are not primary producers as they require organic molecules produced by other organisms for growth; the reduced inorganic sulphur acting as a secondary energy source, facilitating the conservation of organic carbon for synthetic purposes rather than energy metabolism (Tuttle, 1980). The third group are the least understood and are those which are found colonizing the surfaces of both animate and inanimate objects around the hydrothermal vents. It is a bacterium of this type which is found on the inner surfaces of the tubes of the Alvinellid worms and upon their integument, which resembles Beggiatoa, Thiofulvum and Thiothrix sp. and which is probably a species of the latter genus (Tuttle, 1985; Desbruyeres et al., 1985). Chemolithotrophic metabolism by some of the vent-associated bacteria provides then the potential to utilize the geothermal hydrogen sulphide as a source of electrons or reducing power whereby oxidation converts the sulphide to sulphur which in its turn may be oxidised further via sulfite (through an intermediate conversion to thiosulphate and sulphur) to sulphate. Both of these reactions can be coupled directly to the reducton of nicotinamide adenine dinucleotide (NAD) (Jannasch and Wirsen, 1985). This biological oxidation of hydrogen sulphide thus provides the wherewithal for reducing agentmediated chemosynthesis. The direct oxidation of hydrogen sulphide or the cleavage of thiosulphate produces elemental suphur which may form deposits either inside or outside the microbial cell. In either case these can be viewed as a means of energy storage. It has been known for some time that thiobacilli can bind to and oxidise extracellular sulphur at their surface membrane (Trudinger, 1967) and a cytoplasmic, soluble, sulphur-oxidising enzyme has been described (Suzuki, 1974). While the difficulty of culturing the large, filamentous, ThioJix type bacterial associates of the hydrothermal vent polychaetes has impeded elucidation of the details of their sulphur metabolism, their presence within the tubes of Alvinella in large numbers and in close proximity to the deposits of crystalline sulphur is strongly suggestive that they may be responsible for its presence rather than as a product of the worm’s metabolism, for which there is no evidence or precedent elsewhere. Elemental sulphur is, however, hydrophobic and its metabolic production will necessarily present a problem within the aqueous medium of the cytoplasm as it will, effectively, instantly proceed from the monomeric atomic form to a molecular state. Although numerous allotropic forms of sulphur are now known, both from nature and from in vitro manipulation (Meyer, 1976a, b), it might be expected that biologically produced elemental sulphur would be in an amorphous or colloidal form because of its hydrophobic water-insoluble character and tendency to self-association in polar, hydrogen bond-promoting media. Crystalline states of sulphur usually originate from the cooling of melts, from concentration of solutions in solvents more hydrophobic than water, by deposition from the vapour phase or by reactions yielding

Sulphur in hydrothermal vent worms sulphur in special solvent media. The literature prior to the early 1970’s presented a confused view of whether crystalline sulphur can be deposited in largely aqueous media. Mellor’s (1930) and later Gmelin’s (1953) comprehensive surveys of the elements review much of the early literature. Forms of sulphur obtained by acidification of calcium and other polysulphides or sodium thiosulphate with hydrochloric acid, rather than being in the so-called amorphous form, were then considered to be actually in a microcrystalline state. Moeller (1952) did not however consider precipitated forms of sulphur to be crystalline. Interestingly, the so-called colloidal sulphur obtained by passing hydrogen sulphide into sulphur dioxide solution deposits gum-like sulphur on evaporation and this sulphur is reportedly partially soluble in water (Partington, 1951). Since 1960 the unusual molecular complexity of elemental sulphur has become much better understood and the ability of the sulphur atom to bond into a wide variety of ring, helical and linear states is well-documented. Many of these rings display sufficient metastability to exist at room temperatures for appreciable periods, though most have to be generated under conditions which would be considered extreme by biological standards. Cyclohexasulphur is formed in aqueous media, when saturated thiosulphate solutions are acidified with concentrated hydrochloric acid, forming rhomboidal crystals (Meyer, 1976a). It should be remembered, if these conditions are thought of as being biologically extreme, that within membranous organelles enclosed in the cytoplasm the cell is capable of generating both high ionic solute concentrations and extremes of pH (cf. the gastroparietal cells of the stomach or the sulphuric acid-secreting cells of some boring molluscs) by resort to well understood active transport processes. While cyclic forms of sulphur are insoluble in polar liquids, elemental sulphur experiences nucleophilic or electrophilic attack in ionic solutions to form chains of molecular ions and the only likely species that could conceivably occur in a biological situation would be a polysulphide. Polysulphides can be formed in alkaline aqueous media and are stable at neutral pH (Giggenbach, 1974). Cyclic polysulphides incorporating carbon in the rings are known in nature-for example, from the red alga Chondriu californica where, among others, a 12 membered heterocycle contains eight sulphur atoms (Wratton and Faulkner, 1976). The presence of sulphur globules within the large apochlorotic bacteria has long been recognized and is a diagnostic morphological feature of the Thiorhodaceae (Winogradsky, 1887). It was, however, much later that La Rividre (1963) was able to demonstrate for the first time the allotropic state of such inclusions, showing that the sulphur in colonies of Thiovulum majus was of the orthorhombic form. Later Trtiper and Hathaway (1967) showed that the orthorhombic allotrope was also the form that accumulated sulphur takes within marine photosynthetic bacteria. Both of these studies had however involved X-ray diffraction upon sulphur globules isolated from the cells after osmotic shocking and in the dry state. Knowing the ease with which allotropic transitions to

75

the stable orthorhombic state take place it seems likely that this could have happened during isolation. In fact, Hageage et al. (1970) showed that in Chromatium okenii, C. weissei and C. warmingii the sulphur globules, within the living cell or isolated and still in the wet state, were in an unstable allotropic state similar to that found to compose melted liquid sulphur. Although the stable S, ring would seem to be the most likely candidate for the sulphur state in the cellular sulphur globules, Moriarty and Nicholas (1970) conjectured, on the basis of metabolic studies on cell extracts of Thiobacillus concretivorus, using “S enriched sodium sulphide, that a linear form of polymeric sulphur was involved rather than the ring form. This view was based on knowledge that scission of the Sr,ring does not readily occur at room temperature and the experimental observation that the membrane-bound “S intermediate, formed during the enzymic oxidation of the sulphide, was very reactive. It was however conceded that the modifying effects of a lipoprotein environment on reaction rates-given the lipophilicity of elemental sulphur-might mean that the Sr could be more accessible to enzymatic cleavage. Although these latter authors deduced the involvement of a lipoprotein membrane with the elemental sulphur within the thiobacillal cell this seems to have been based largely upon the observation of a primarily hydrocarbon background to the sulphur mass spectra of the ‘membrane-bound’ sulphur. Schmidt and Kamen (1970) also noted a thin ‘membrane’ surrounding the sulphur in sections of Chromatium and electron microscope observations by Nicolson and Schmidt (1971) led to the conclusion that this membrane, rather than being the usual trilaminar lipid-protein complex, in the accepted sense of a cellular membrane, was in fact an entirely proteinaceous structure. Schmidt et al. (1971) confirmed this finding and, on the basis of unpublished inhibition studies, rejected the notion that the protein had an active role in sulphur metabolism speculating instead that the ‘membrane’ might provide the means of isolation of the elemental sulphur from the cytoplasm. The balance of hydrophilic and hydrophobic amino acid species in the protein would not however have led to the conclusion that it would provide a hydrophobic barrier. The tubes of Afvinellu are not the only situation in the hydrothermal vent fauna1 community where elemental sulphur either as globules or crystals occurs. Both Jones (1981) and Rau (1981) comment on the presence of crystals or particles of pure elemental sulphur in the trophosomal tissue of the vestimentiferan worm Riftiu. Although the trophosome is the extensively lobulated structure which, with the gonads and gonoducts and an extensive blood vessel system, occupies the trunk cavity of this gutless worm, most of its ‘tissue’ is in fact composed of large numbers of cells filled with densely-packed sulphidemetabolizing bacteria in endosymbiotic relationship (Felbeck and Somero, 1982; Cavanaugh, 1985; Felbeck et al., 1985). Although Jones (1981) reports these cellular sulphur deposits as being crystalline one cannot be certain, in the light of what has already been said, that this is not an allotropic phase change

76

!hPHEN HUNT

in consequence of the techniques used to prepare the tissue for microscopy. Several species of bivalve moiiuscs dwelling in sulphide-rich environments, and including Lucinoma unnulata, Calyptogena efongata and Lucina jfuoria’ana, and the hydrothermal vent species C. magniJca (Chiidress et al., 1987), also maintain a symbiotic realtionship with sulphur-metabolizing bacteria. Here the bacteria are found in the gill tissue occupying ‘bacteriocyte’ ceils in a layer immediately beneath the ciliated ceil layer (Vetter, 1985). This places them in ceils close to the gas exchange interface. These tissues contain sulphur particle inclusions. Jean Vovelie, of the Universitk Pierre et Marie Curie, in a personal communi~tion of unpublished results, has shown me pictures, taken by Fiaia-Medioni at the Banyuis Marine Station, of rhomboidal suiphur crystals within a gill filament of the hydrothe~ai vent species Cafyptogena phaseoliformis. Vetter (1985) observed both birefringent crystals and birefringent globules of suiphur within the peripiasmic space of the bacterial

ceils of the non-vent species listed above but noted that as frozen sections dried out, more crystals appeared and the globules underwent a phase transition to solid crystals. Tissue smears of living gill tissue in deoxygenated buffer contained only round suiphur globules and these could be prepared in that state by density gradient centrifugation and shown to undergo the transition to crystal form on drying. Sulphur inclusions are not found in the cytoplasm of the moiiuscan ceils with the exception of the contents of some lysosome-like organeiies which also contain bacterial fragments. Vetter (1985) believed the globules to be suiphur in liquid crystal form and commented upon the loss of Maltese cross patterns, in polarized light, as the phase transition to a solid crystal took place. Nicolson and Schmidt (1971) and Schmidt et ai. (1971) noted myeiin figure formation as the suiphur spherules in Chromatium species ruptured-another characteristic of liquid crystals. Vetter (1985) discounts the possibility that sulphur formation from sufphide in the gill is a detoxification mechanism to protect the animal tissue since the tissue contains virtually no elemental suiphur but considers the deposits to represent energy stores permitting the symbiotic bacteria to continue functioning when external suiphide concentrations fail temporarily. A close relative of Afuineila is another vent associated polychaete Paralvinella. Paralvinella has no tube but secretes a copious giycoprotein mucin, probably for protection (Taimont and Fournet, 1990). The fresh mucous coat is coiouriess or whiteish but rapidly becomes bright yellow with deposits of amorphous and iobateiy globular sulphur and later mineralizes with sulphides of iron and copper suiphides and strontium sulphate. In this case only so far is there some doubt about a bacterial involvement in suiphur deposition. Juniper et al. (1986) have not found an epibiotic flora, comparable to that of Alvinella, associated with the body surface of this worm though carbon dioxide fixation is demonstrable in ail regions of the body. However, filamentous bacteria, which are probably chemosynthetic, have been found in the gut, suggesting at least a potential route for suiphide

ingestion, auto~op~c chemos~thesis and subsequent export of elemental suiphur. In the case also of the Alvinella-associated filamentous bacteria, globules of elemental sulphur are quite commonly observed within the cells though these can disappear in response to a fail in external hydrogen suiphide tension thus suggesting a drawing upon the internal So store for oxidative meta~lism (Nelson and Jannasch, 1984; Prieur and Jeanthon, 1987; Gail1 and Hunt, 1991). Indeed the likely explanation as to why Voveiie and Gail1 (1986) did not find sulphur in their tubes from Alvinella may lie in harvesting animals from a field temporarily deprived of sulphideenriched hydrothermal waters. If this is the case then one may draw concIusions for the converse case where the external hydrogen suiphide concentration is so high as to demand export of excess sulphur to be deposited as crystals, partly as a means of capitaiizing on a rich energy source while avoiding the problem of overloading storage sites internally, and partly perhaps as a detoxification mechanism. Aiternatively the death of bacteria containing high concentrations of segregated inorganic elemental suiphur might also result in a transformation of the latter into crystalline deposits. The presence of stable liquid rather than solid crystals of suiphur within the ceils of bacteria associated with the hydrothermal vent animals becomes more easy to understand in the light of knowledge of the molecular state of sulphur. There is considerable torsion in the S-S-3-S structure, and the various ring forms of octa suiphur as well as the potential for linear and helical forms ail contain in their asymmetry and large size the propensity for liquid crystaiiinity. If only a little water is present to interfere with Van der Waal’s suiphur-sulphur bonded interactions between asymmetric molecular units, then stable liquid crystals will be a consequence of suiphur secretion within ceils with a high pro~bility of choiesteric structure and the development of myelin figures. Water loss after release from ceils would then consequentially cause the transition to the more stable solid crystal state as asymmetric rod-like moiecuiar suiphur units became able to form bonded interactions with one another. REFERENCES

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