TRACE METAL CHEMISTRY AND SILICIFICATION OF MICRO-ORGANISMS GEOTHERMAL SINTER, TAUPO VOLCANIC ZONE, NEW ZEALAND

K. L. BROWN’, E. J. S. L. K. A. CAMPBELL’

‘Geology University of Auckland of Geology, Portland State University, Portland, Oregon, USA

SUMMARY part of a pilot study investigating the role of microorganisms in the of As, Sb, and Hg, the inorganic geochemistry of seven different active sinter deposits and their contact fluids were characterised. The extractions showed whether metals were loosely exchangeable or bound to carbonate, oxide, organic or crystalline fractions. Hyperthermophilic microbial communities associated with sinters deposited from high temperature (92-94°C) fluids at a variety of geothermal sources were investigated using SEM. The rapidity and style of silicificationof the can be correlated with the dissolved silica content of the fluid. There was little evidence to suggest that any of the heavy metals were associated preferentially with the hyperthermophiles at the high temperature ends of the terrestrial thermal spring ecosystems studied.

1.0 INTRODUCTION rates, pre-sterilised glass microscope slides were deployed as artificial substrates at some sites. Arsenic, antimony, boron, thallium and mercury Bulk concentrations of trace elements were (As, Sb, B, Hg) are commonly found analysed semi-quantitatively using X-ray concentrated in silica sinter deposited around fluorescence spectrometry. Bulk samples natural New Zealand hot springs (Weissberg, also were analysed to determine whether the trace and in the sulphide scales of geothermal metals in the sinters were exchangeable, or bound pipelines of some power stations of the Taupo to oxides, carbonates, organics, or incorporated Volcanic Zone (Brown and McDowell, 1983). into the crystalline fractions, by using a series of Release of these elements from geothermal waters, sequentialextractionsoriginally developed for soil steam condensate and leachate into waterways can analysis by Tessier et al. with pose an environmental risk. Investigation of the modifications by Kim and Fergusson (1991). manner in which these elements are Biological samples for SEM were preserved in in silica sinter may reveal an avenue of gluteraldehyde, then rinsed twice in distilled remediation where elements such water, subjected to an ethanol dehydration series, I as these cause contamination. The surfaces and and critical point dried to prevent volume loss extracellular components of bacteria are highly (Ruffolo, 1974). reactive and they could provide ideal sorption surfaces for the nucleation of inorganic phases, 2.2 DNA preparation however, so far, the interactions that might occur between metals and microbiota surfaces have not To investigate whether DNA could be extracted been extensivelystudied. fiom vitreous sinter, geyserite fkom Tokaanu was subjected to molecular analysis. The presence of 2.0 SAMPLE SITES METHODS bacterial DNA in the sample was investigated by i polymerase chain reaction (PCR) amplification of The sampling sites comprised the natural hot a 200 base pair of the small springs at Waikite (WKT), man-made ribosomal RNA gene, using Bacterial-specific environments such as primers. Samples with Bacterial primer were and weirboxes at the placed in wells in Fig. 2F. included Wairakei Geothermal Power Station and the a positive control to ensure that the amplification Ohaaki Pool (OHK), and an abandoned was properly (wells 5 and a blank of geothermal bore at Tokaanu (TK). distilled water as a contamination check (well 7), and a ‘ladder’ to ensure complete sample 2.1 Analyses migration (well 8). PCR products were separated by gel electrophoresis and identified under W The contact fluids were analysed for As, Sb, B, illumination. and Hg by ICP-MS. In order to qualitatively assess sinter and microbial biofilm accumulation

39 Table 1 Chemistry ofgeothermalwaters (allvalues in below detection limits)

3.0 RESULTS low Fe concentrations had low As concentrations. It was also noted that when the arsenic and iron 3.1 Fluids concentrations antimony concentrations increased. Results for trace metal concentrations in geothermal fluids analysed by ICP-MS are shown 3.2 Fluid-sinter ratios in Table 1. The silica concentrations in the fluid collected the geothermal power To determine whether As, Sb, B, Tl and Hg were developments were considerably greater than selectively concentrated in sinter, the trace metal those of the natural features. Except for the concentrations in the fluid and sinter phases were Waikite site, the water samples display reasonably normalised to the silica concentration of each Uniform concentrationsof B, As,Hg, Sb, and Tl. sample, in the Waikite and Hg in the Tokaanu geyserite and Flash plant 11 sinter were 3.2 Sinter the only trace metals that were selectively concentrated in the sinter. Results for trace metal concentrations in the bulk sinter, as analysed by show that except for 3.3 Selectiveleaching the and the Tokaanu vent geyserite samples, the silica concentrations are all greater The sequential extraction analyses showed that As than 80%. The Waikite sample is a mixture of does not exhibit preferential association with any silica and calcite. Among the trace metals, only of the extractable Sb is bound As and Sb were found to be above the detection preferentially with the crystalline of each limits for However, total concentrations of of the sinters. Although B is predominantly all of the trace metals were calculated during the associated with the crystalline there are sequential extraction analysis (Table 2). High significant quantities associated with some of the iron concentrations were found in the Tokaanu other extracted especially at Ohaaki vent geyserite, which also contained the highest where it is found in anomalous concentrations in arsenic concentration. The antimony all of the relative to the other sinter concentrations are reasonably Uniform for samples. Tl occurs variably in all except samples. the exchangeable The highest concentrations of Tl are associated with the After this study began, water draining from an organic fraction in the high temperature acid iron-rich natural geothermal was geyserite Tokaanu. The only significant diverted away the Wairakei main borefield concentrations of Hg occur primarily with the drain consequently, two sinter organic fraction from the moderate temperature samples from this site were one taken silica oncoids at from the drain before, and one taken after the diversion. The showed that the sample with high Fe concentrations also had elevated As concentrations, while the sample with

40 Table 2 Trace element total concentrations in the sinters calculatedfrom the sum of the sequential extractions. A single total (nonsequential) digest of TKI is also shown (ppm) = below detection limits)

Element Sb As B TI TKI 269 1210 314 0.47 6.6 TKI single 504 2360 458 0.3 9.2 digest 150 39 346 0.01 1.3 TK3 198 214 622 3.88 OHK 138 26 1703 2.1 WKFPII 116 IO 91 0.07 3.0 16 93 7 73 WKMD 68 1237 258 0.05 1.5 WKEB 105 24 195 0.01 1.6 WKT 35 85 479 0.02 0.5

3.4 Microbiota subaqueous geyserite, the hyperthermophiles that colonise Flash Plant 11 subaqueous sinter were In this study we have grouped the microbial often extensively and display a range of communities investigated according to their apparent diameters and lengths as shown in Fig. environmental niche growthhabitats described by Golubic et al. (1981) as epilithic (on upper surface), hypolithic (underneath), and endolithic Figs 2A-2D show an example of a hypolithic We therefore identified the microbial community that colonised the underside of a communities associated with the outennost columnar geyserite knob Tokaanu. Prior to accretionary surfaces as epilithic, and the collection, the knob was communities associated with the undersides of directly to periodic splashes of hydrothermal fluid sinter as hypolithic. ejected the vent SEM investigation revealed that the hypolithic thatgrow attached to the community consists primarily of organisms upper- or outermost of sinters may be by two different morphotypes. One located either in subaerial, subaqueous, or mixed morphotype is comprised of long rods in regimes. length and in width. The other Figs. and show examples of the epilithic morphotype is by long filaments that communities that colonised the reach up to or more in length and subaqueous geyserite Tokaanu. Prior to in width. The of a thin, at least collection, the Tokaanu geyserite sample was silicified microbial biofilm matrix is shown in continuallysubmerged in water with a of Fig. 2E. Molecular analysis of taken 7.7. Hyperthermophilic microorganisms that the surface on the underside of the Tokaanu adhere to the geyserite surface, or to each other, columnar geyserite shows the presence of display a similar morphotype that consists of long bacterial DNA (Fig. 2E). of the thin filaments. Shown in gene a 200 base pairs long, Fig. is an example of the filamentous typical of bacterial DNA. hyperthermophiles diameter) that attached to a glass slide deployed at the Endolithic phototrophic microorganismswere also effluent of Flash Plant 11 located at the observed to occur within the outermost few Geothermal Power Station. The glass slide was millimetres of columnar geyserite at Tokaanu. continuously submerged in water = 8.1) Green and orange pigments of the phototrophic for two weeks. The epilithic community that endoliths visible in views of the subaqueous sinter Surfaces at Flash the sinter. Within a few millimetres of the Plant 11, and the effluent at the Tokaanu outennost geyserite surface, we found endoliths , live at approximately the same exposed on a of the temperature. However, in contrast to the Tokaanu Tokaanu geyserite and this epilithic hyperthermophilic community on the endolithicmicrobial community autofluoresces.

41 I

Figure SEM photomicrographs of communitiescollected 11

a

Figure 2. Geyserite samples collected Healy's Bore 2, Tokaanu at magnification(A through E); DNA fiomthe columnar geyserite collected at Tokaanu.

42 4. DISCUSSION ppm), which are less than half that of the fluid at Flash Plant 11 at Wairakei (547 ppm). These 4.1 Microbes and silica observations illustrate that in the ecosystems by hot springs have been recognised for fluids with supersaturated dissolved silica decades as ideal ecosystems for the concentrations, the formation of heavily silicified preservation of thermophilic microorganisms microfossils is likely to be a (Walter, 1976; Cady and Fanner, 1996; Renaut et occurrence. al., Cady and co-workers (Cady et al., 1995; Cady and Farmer, 1996) only recently We also have observed that some of the demonstrated that the biofilms hypolithic found on the of a associated with geyserite also can become columnar geyserite collected in the splash zone at preserved in these environments. Bore 2 at Tokaanu display the earliest stages of a type of silicification (Fig. By high and low silica concentration Inside nearly of the cavities of the silicified environments, we have found that the rapidity honeycomb structure shown in Fig. 2B are short with which epilithic hypolithic filaments, most of which display communities become silicified electron contrast that indicates they are not be correlated with the concentration of silicified. However, as shown in Fig. 2D, one dissolved silica in the hydrothermal fluids. cavity contains short filaments inside which have electron dense silica colloids As shown in Fig. 1, various morphotypes of diameter). hyperthermophiles can be found in the geyser vent at Tokaanu (Figs. and and around the effluent at Flash Plant 1 Wairakei (Figs. 1 and 20 A The hyperthermophiles associated with the epilithic on the geyserite specimen shown in Figs. and do not appear to be Evidence that this hyperthermophilic community is not extensively silicified is surmised the presence of the remains of the which appears as a amorphous substance associated with the geyserite and interspersed amongst the It is worth noting that silica did not precipitate onto glass slides deployed for two weeks in the geyser effluent at Tokaanu. In contrast to the microbes observed fiom the vent at Tokaanu, the hyperthermophiles collected fiom a glass slide deployed for two weeks at Flash Plant 11 (Figs. 1C-D) display various degrees of silicification. Fig. demonstrates that cell are approximately the same size even though they occur as collapsed, or almost entirely entombed in silica colloids diameter). Fig. reveals that the hyperthermophiles associated with the sinter deposit at Flash Plant 11 can be heavily silicified, and characterised by a range of apparent Although long and short filaments can Figure 3. Cryptoendolithic community in sinter be seen in this sample, it is impossible such Tokaanu A) The mow shows the position an image to determine the true of a pigmented zone. Stratified diversity of the hyperthermophilic autofluorescentfilaments visible in a thin section. members. Merging and agglomeration of silica with colloids time, and continued silica A stratified community occurs in deposition, produced filament-type objects of the layers of silica a columnar different sizes. However, size analysis of geyserite collected in the splash zone at Tokaanu several non-silicified revealed only one (Fig 3). This is superficially similar to morphotype attached to the sinter surfaces in the community of viable populations. Decreased silicification of that as a rind in surface microbes and the of sinter deposition at the dry valleys of Johnston, Tokaanu is consistent with the lower silica 1989). Silica associated with the geothermal concentrations in the waters at that location (287 is most porous at its outer

43 where the secondary porosity of the silica has not the Tokaanu vent geyserite sample. However, yet been occluded to form a dense, impenetrable deposit as it does inside the geyserite. The 2000) detected localised concentrations of phototrophic endoliths are located at similar and in the sinters. It is depths in the interior of the two different types of possible that additional, as yet unidentified, deposits: for the geyserite and mechanism may be responsible for As for the Antarctic sandstone. The Tokaanu concentrationat Tokaanu. geyserite endoliths are also similar to the Antarctic endoliths in having blue-green and black Sb increased fkom zero to 132 ppm when Fe and pigments. As decreased. It is not why there is an inverse relationship between As and Sb Fig. 3B shows discrete layered with concentrations. analysis of powdered sinter vertically oriented filaments and that indicates that the primary silica phase is contain very few autofluorescing filaments. submicroscopic-size While a stratified endolithic community clearly silica particles would not be by occupies the outermost millimetres of the and may for the concentration of some Tokaanu columnar geyserite, the presence of trace metals. It is worth noting that microscopic numerous, strongly pigmented (especially dark Sb-As-Mg-S crystals were observed to occur in green) layers in the of the geyserite the geyserite sample Tokaanu. suggests another possible explanation for the phototrophic filaments in some of the columnar Boron geyserite laminations. If periods of time occur From the sequential extraction analysis it appears when fluid flow from the geyser effluent wanes, it that the majority of the boron is bound in the is likely that phototrophic biofilm consisting of crystalline of the sinter. Given the motile cyanobacteria could develop on the upper tendency of boron to replace silicon in silicates of the geyserite. Communities of viable (Grew and it is possible that a phototrophic cyanobacteria were observed in considerable proportion of boron could exist topographic lows around the geyserite columns within submicroscopic-size silica phases in the where splashed water accumulates and cools rapidly. At a later time, if the effluent activity it is likely that the phototrophic Mercury and Thallium community would rapidly become in Selective extraction analyses of the seven different silica as geyserite accretion resumed. Prior to the sinter deposits revealed that Hg and Tl are the time the cyanobacteriapigments they could- only trace metals found to be associated primarily be visible in thin sections of the geyserite, and with the organic Tokaanu. The give the of ’endolithic’layers. relative fluid-sinter concentration ratios of Tl and Hg in water samples these localities also 4.2 Sequential extractions were to be high. The sample fiom Waikite is unusual in that there is no detectable The series of sequential extractions was obtained associated with the carbonate or organic sites, for the suite of trace elements in order to despite this sinter consisting of 50% calcite. In a how the As, Sb, B, and Hg artificial substrate experiment at Waikite, chemically bound in the sinter. The trace element no were found to colonise the concentrations obtained by summing the over a two week period. These concentrations measured at each sequential observations were consistent with the absence of extraction provide the only determination of Hg, trace metals associated with the for the and B since all three elements were present in organic fkom The organic the sinter in concentrations below the detection may originate either sulphide limit of analysis. or the thermophilic biomass, however total organic carbon comprises less than 0.5% of Arsenic and Antimony the sinter mass. This does not preclude the results for As are in agreement with the data possibility of microbial involvement in the obtained by sequential extractions of geothermal sequestering of these elements, as mass sinter undertaken by et al. calculations do not take interactions into which showed that As was dispersed and microorganisms and their among the in silica sinter. matrices have extremely large Finlayson and Webster (1989) and (1996)have established the of As 5.0 CONCLUSIONS with silica and Fe oxides. This study confirms that As precipitated in the sinter declined Silica concentration in geothermal fluids 938 ppm to 11 ppm when precipitated Fe the style and rapidity with which decreased 7410 ppm to 1080ppm. This ‘iron are silicified. effect’ could explain the high concentrationsin

44 It is difficult to distinguish endolithic fkom Cady, S.L. and Farmer, J.D., 1996. Fossilization epilithic microorganisms in dynamic near-vent processes in siliceous thermal springs: trends in environments because of the rapidity of preservation along thermal gradients. In: G.R. silicification and likely changes in flow Bock, J. A. (Editor), Symposium on characteristicsof the . Evolution of Hydrothermal Ecosystems on Earth (and ?). Ciba Foundation Symposium. - Sb will preferentially precipitate with silica Wiley, Ciba Foundation, London, pp. 150-173. (although the mechanism is unknown) when there is no Fe present, and As will not significantly Finlayson, J.B. and Webster, J.G., 1989. precipitate with silica unless the Fe is present. Relationship Between Arsenic, Iron and Silica in the Main Drain of the Wairakei Borefield, DSZR - Hg and were preferentially associatedwith the TechnicalNote 7. organic hction in some of the sinters studied. Some studies Gadd, 1988; Poole and Gadd, Gadd, G.M., 1988. Accumulation of Metals by 1989; Brierley, 1990; Ferris, 1990; Mann, 1990; Microorganisms and Algae. In: H.J. Rehm and Beveridge, 1994; Beveridge, 1995; (Editor), Special Microbial Processes. Schultze-Lam et al., Konhauser and Ferris, Biotechnology. VCH, pp. 40 1996) have shown that heavy metals are associated preferentially with microbes. For the Golubic, S., I. and Schneider, J., hot springs investigated in this study, the 1981. The lithobiontic ecological niche, with preferential adsorption of heavy metals other than special reference to microorganisms. Journal of mercury or thallium by hyperthermophilic Sedimentary Petrology, microorganisms was not commonly observed in high-temperature siliceoussinters. Grew, E.S. and Anovitz, L.M. (Editors), 1996. Boron: mineralogy, petrology and geochemistry. 6.0 ACKNOWLEDGEMENTS Reviews in Mineralogy, 33. Mineralogical Society of America,, Washington, 862 pp. Many thanks to the Foundation for Research, Science, and Technology, for funding the project; Johnston, C.G., 1989. Distribution of Inorganic Kerry Webster, Department of Species in Two Antarctic Cryptoendolithic Conservation, Contact Energy, and Sue Turner Microbial Communities. (Biological Sciences, University of Auckland). Journal, 137-154. We also acknowledge the of the Tokaanu area Ngati for field access Kim, N.D. and Fergusson, J.E., 1991. permission. Effectiveness of a commonly used sequential extraction technique in determining the speciation 7.0 REFERENCES of cadmium in soils. Science of the Total 105: 191-209. Beveridge, T.J., 1995. Sorption of Metals on Bacterial Surfaces. In: L. W. D, Konhauser, K.O.and Fems, F.G., 1996. Diversity Rajan, S. (Editor), Biominet: Eleventh Annual of iron and silica precipitation by microbial mats Meeting Proceedings. Natural Resources Canada, in hydrothermal waters, Iceland: Implications for Canada, pp. 1-10. Precambrian iron formations. Geology, 24: 326. Brierley, C.L., 1990. Metal Immobilization Using Bacteria. In: H.L.Ehrlich, Brierley, C. L. (Editor), Mann, H., Biosorption of Heavy Metals by Microbial Recovery. Environmental Bacterial Biomass. In: B. (Editor), Biotechnology. McGraw-Hill Publishing Co., Biosorption of Heavy Metals. Press, Boca USA, 93-137.

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