ecological assessment and geological significance of ...dzumenvis.nic.in/microbes and metals...

Geomicrobiology Journal, 18:259–274, 2001Copyright C° 2001 Taylor & Francis0149-0451/01 $12.00 + .00

Ecological Assessment and Geological Signi� canceof Microbial Communities from Cesspool Cave,

Virginia

ANNETTE SUMMERS ENGEL

Department of Geological SciencesUniversity of Texas at AustinAustin, Texas, USA

MEGAN L. PORTER

Department of ZoologyBrigham Young UniversityProvo, Utah, USA

BRIAN K. KINKLETHOMAS C. KANE

Department of Biological SciencesUniversity of CincinnatiCincinnati, Ohio, USA

Microbial mats from hydrogen sul�de-rich waters and cave-wall bio� lms were inves-tigated from Cesspool Cave, Virginia, to determine community composition and po-tential geomicrobiological functioning of acid-producing bacteria. Rates of microbialmat chemoautotrophic productivity were estimated using [14C]-bicarbonate incorpo-rations and microbial heterotrophy was determined using [14C]-leucine incubations.Chemoautotrophic� xation was measuredat 30.4 § 12.0 ng C mg dry wt¡1h¡1, whereasheterotrophic productivity was signi�cantly less at 0.17 § 0.02 ng C mg dry wt¡1h¡1.The carbon to nitrogen ratios of the microbial mats averaged 13.5, indicating that themats are not a high quality food source for higher trophic levels. Ribosomal RNA-basedmethods were used to examine bacterial diversity in the microbial mats, revealing thepresence of at least � ve strains of bacteria. The identity of some of the strains couldbe resolved to the genus Thiothrix and the Flexibacter–Cytophaga–Bacteriodesphylum,and the identity of the remaining strains was to either the Helicobacter or Thiovulumgroup. Two of 10 sulfur-oxidizing,chemoautotrophicpure cultures of Thiobacillus spp.(syn. Thiomonas gen. nov.) demonstratedthe ability to corrode calcium carbonate,sug-gesting that the colonizationand metabolic activity of these bacteria may be enhancingcave enlargement.

Received 1 July 2000; accepted 1 March 2001.This research was partially funded by research grants from the National Speleological Society, the Geolog-

ical Society of America, the American Association of Petroleum Geologists, and the University of Cincinnati.Permission to sample in Cesspool Cave was granted by its landowners and special thanks are extended to them.The authors also thank R. Popa, V. Shaprio, and E. Waits for help with laboratory and � eld work, and D. Hubbardfor information regarding the cave.

Address correspondence to A. S. Engel, Department of Geological Sciences, The University of Texas atAustin, C1140, Austin, TX 78712, USA. E-mail: [email protected]

259

260 A. S. Engel et al.

Keywords cave, chemoautotrophy,microbial mats, primary productivity, speleogen-esis, sulfur bacteria, Thiobacillus, Thiomonas, Thiothrix

Cycling of sulfur compounds by bacteria and subsequent biogeochemical interactions inmany subsurface environmentshave been well studied, especially from geothermal systems(Brock and Mosser 1975;Deming and Baross 1993;Moyer, Dobbs, and Karl 1995) and acidmine drainage (Edwards, Bond, Gihring, and Ban� eld 2000;Harrison 1984; Johnson1998).Reduced sulfur compounds that are generated throughabiotic or microbial sulfate reductioncan be used as energy sources by sulfur-oxidizing microbial populations, often generatingsulfuric acid. Consequently, sulfur-oxidizing bacteria can cause destruction of concretein buildings and sewer pipes (Milde, Sand, Wolff, and Bock 1983; Parker and Jackson1965; Zherebyateva, Lebedeva, and Karavaiko 1991), granite deterioration (Wagner andSchwartz 1967), and have been studied in association with weathering of volcanic rocksin Yellowstone National Park, Wyoming (Brock and Mosser 1975). Sulfur cycling andmicrobiallygenerated sulfuric acid may also enhance carbonate rock dissolutionand caverndevelopment through the process of sulfuric acid speleogenesis (Brigmon, Martin, Morris,Bitton, and Zam 1994; Hill 1996; Hose, Palmer, Palmer, Northup, Boston, and Duchene2000; Hubbard, Herman, and Bell 1986; Lowe and Gunn 1995; Sarbu 1996; Smyk andDrzal 1964; Vlasceanu, Sarbu, Engel, and Kinkle 2000).

Since the discovery of the chemoautotrophically based groundwater ecosystem inMovile Cave, Romania (Sarbu and Popa 1992; Sarbu, Kane, and Kinkle 1996), there havebeen numerous studies of other sulfur-based microbial communities in caves and similarsubsurface environments, including the submarine caves at Cape Palinuro, Italy (Mattison,Abbiati, Dando, Fitzsimons, Pratt, Southward, and Southward 1998), meromictic ponds incenotes of the Yucatan Peninsula, Mexico (Wilson and Morris 1994), Cueva de Villa Luz,Mexico (Hose et al. 2000), and Cae Coch mine, North Wales (Johnson 1998). There haveonly been a few studies, such as those from the Sulphur River of Parker Cave, Kentucky(Angert, Northup, Reysenbach,Peek, Geobel, and Pace 1998), and the Frasassi Caves, Italy(Vlasceanu et al. 2000), that address potential geomicrobiologicalconsequences of micro-bial sulfur oxidation, in particular the enhanced dissolution of limestone and accumulationof secondary mineral deposits within caves.

The pioneering works of Hubbard et al. (1986, 1990) in Cesspool Cave, Virginia,suggest that sulfur-oxidizing bacteria were contributing to cave formation by generatingsulfuric acid. They based their hypothesison the presence of the morphologicallyconspicu-ous sulfur bacteria: Beggiatoa, Thiothrix, and Achromatium. The present study extends thiswork by examining the sulfur-based microbial community and its impact on speleologicalprocesses in Cesspool Cave. Microscopic examinations were made of the microbial matsand associated substrates from the cave. Microbial chemoautotrophic and heterotrophicproductivity measurements were used to evaluate metabolic activities, and analysis of matcarbon and nitrogen content was done to assess the potential ecological impact of sulfur-based metabolism in Cesspool Cave. The bacterial community was evaluated using 16SrRNA-based molecular techniquesand sulfur-oxidizingbacteria were cultured. In addition,isolated strains were tested for acid production and microbially enhanced acid dissolutionof the cave was considered.

Materials and Methods

Study Location

Cesspool Cave is a small (<20 m of passage) cave developed in a travertine-marl complexof Quaternary age located along the Sweet Springs Creek in Allegheny County, Virginia

Microbial Communities from Cesspool Cave 261

FIGURE 1 Plan-view map of Cesspool Cave, Allegheny County, Virginia, modi� ed fromHubbard et al. (1990). Shaded regions refer to � oor substrate or water. Water, mat, andsediment samples were taken from the spring and locations 1–3, and wall mats were takenfrom location 4.

(Hubbard et al. 1990;Figure 1). A sul� dic (0.45 to 0.66 mM), nonthermal (12–13±C) springdischarges into a back pool, � ows out of the cave, and resurges into a surface pond. Thehydrogen sul� de is thought to originate from oil� eld brine solutions that � ow up-dip alonglocal faults (Hubbard et al. 1990). Some incidental sunlight reaches the back pool area dueto the limited passage length and large surface entrance. Gypsum crusts form on the walls ofthe back room, with a patchy distribution covering approximately 30% of the wall surfaceabove the pool, and reach up to 1 cm in thickness where they occur.

Sample Collection

Water samples were collected from the spring, pools, and resurgence of the cave (Figure 1)to supplement detailed chemical analyses by Hubbard et al. (1990). Samples of microbialmats and bio� lms were taken from the cave pools, resurgence area, and wall crusts usingsterile forceps and tubes. The samples were placed on ice for transport to the laboratory,transferred to sterile screw-cap tubes containing 50% (v/v) glycerol, and stored at ¡80±C.

Electron Microscopy

Microbial mats were processed for scanning electron microscopy (SEM) by � xing biolog-ical material using a chemical critical-point drying method modi� ed from Nation (1983).Samples were � xed for 4 h with 25% gluteraldehyde, then dehydrated with a series ofethanol washes. Air-dried samples were mounted on aluminum stubs, sputter-coated withgold, and examined using a JEOL JSM-T330A SEM at the University of Texas at Austinusing 30 kV for an accelerating voltage.


Community Productivity Studies

In the � eld, microbial mats were collected aseptically from the spring area and transferredto a 50-ml sterile bottle. The mats were shaken vigorously to homogenize them and 2-mlaliquotswere transferred to 20-ml scintillationvials containing10 ml of cave water. Parallelexperimentswere run with two radiolabels (ICN Pharmaceuticals,Costa Mesa, California).Each mat sample received either 0.2–1 ¹Ci of [14C]-sodium bicarbonate (56 mCi/mmol) or0.2 ¹Ci of [U-14C]-leucine (320 mCi/mmol). The high concentrations of leucine (67nM)were used to minimize intracellular dilution (Jørgensen 1992). Two replicate samples wereincubated for each radiolabel for 15, 30, and 60 min. At the end of each time period, incu-bations were stopped with the addition of 4% (� nal concentration) formalin. Incubations,with � xative added before the radiolabel, were used as negative controls. All samples weretransported to the laboratory for biomass and radiolabel counting.

Subsamples from each replicate were � ltered using preweighed nitrocellulose � lters(0.2 ¹m pore size) and washed with water. Bicarbonate incubationswere also washed with0.1 M HCl and leucine samples were washed with cold trichloroacetic acid to remove anyexcess radiolabel. Filtered samples were then dried, weighed, dissolved using NCS tissuesolubilizer, suspended in scintillation� uid (Fischer), and measured using a Packard 2200caTri-carb scintillation analyzer (Meriden, Connecticut).

Productivity rates for each radiolabel were determined from the total counts incor-porated, minus the negative control activity, and standardized using the mg dry weight(mgdw) of each sample. Rates of primary productivity in terms of 12C were calculatedusing the methods of Wetzel and Likens (1991), whereas heterotrophicbiomass productionwas estimated using a conversion factor of 3.1 mg C mol¡1 leucine incorporated (Kirchman1993).

Carbon and Nitrogen Analysis

The content of carbon and nitrogen in the microbial mats was analyzed from pool andwall mat samples. The mats were desiccated, exposed to HCl fumes to remove residualinorganic carbonates, and analyzed for carbon and nitrogen in a Perkin-Elmer Series IICHNS/O Analyzer 2400 (Norwalk, Connecticut). Six replicates for each sample were used.

Culturing and Acid Characterization of Sulfur-oxidizing Bacteria

Microbial mats collected in the � eld were transferred to liquid media for culture en-richments of chemoautotrophic, sulfur-oxidizing bacteria, using methods modi� ed fromBrierley (1966) and Kuenen, Robertson, and Tuovinen (1992). Enrichments were done us-ing thiosulfate at pH 6 (medium MS), and elemental sulfur at pH 4 (medium S1) andpH 6 (medium S2). Liquid cultures were examined after 1 week incubation at 28±C,but were maintained for 3 months to isolate slow-growing strains. Once bacteria wereobserved microscopically in the liquid cultures, they were plated on MS or S2 mediacontaining 2% Noble agar. After 5 to 10 days, individual colonies were randomly iso-lated by streak-plating onto new plates and subsequent re-inoculation into liquid medium.Isolates were subdivided on the basis of morphology, as determined by phase-contrastmicroscopy.

Changes in media pH were used to detect acid production by the microorganisms andisolated strains were subsequently classi� ed as acid-producing and nonacid-producing byplating on media containing 0.01 g bromocresol green (BG). Colonies that produced acidon BG plates were also tested for calcium carbonate (CaCO3) dissolution, using 2.5 g ofsterilized CaCO3 added to individual MS or S2 plates.


DNA Extraction

Total genomicDNA was extractedfrom environmentalsamples of poolmats or pure culturesusing cell lysis methods modi� ed from Zhou, Bruns, and Tiedje (1996) and manufacturerguidelines for Puregene DNA extraction kits (Minneapolis, Minnesota). The followingmodi� cations were made: 1 ml of sample was used, samples were incubated at 37±C for5 min prior to the addition of 20 ¹l/ml of sodium dodecyl sulfate, a freeze– thaw serieswas used and was followed by incubation at 65±C for up to 2 h, and nucleic acids wereprecipitated in isopropanol for 45 min on ice. DNA was puri� ed using Sepharose 4B spincolumns (Jackson, Harper, Willoughby,Roden, and Churchill 1997) or following manufac-turer guidelines for WizardPrep DNA cleaning kits (Promega, Madison, Wisconsin). ThreeDNA extractions were done and combined for each sample analysis.

Polymerase Chain Reaction (PCR) Ampli� cation

Nearly full length 16S rRNA (rDNA) gene sequences were ampli� ed using primer pairs(8F/1492R) for bacteria, as described by Lane, Pace, Olsen, Stahl, Sogin, and Pace (1985). Anegativecontrol tube containinga sterile water solution instead of DNA was used. The PCRprocedure was conducted in a Perkin-Elmer 480 thermal cycler (Norwalk, Connecticut) for30 cycles under the following conditions: denaturation at 95±C for 50 s, primer annealingat 48±C for 45 s, chain extension at 72±C for 100 s. PCR products were examined on 0.7%agarose gels.

Cloning of 16S rRNA Genes

The cloning and transformation procedure was performed using the TA Cloning Kit (In-vitrogen, Carlsbad, California) per manufacturer recommendations. Two cloning reactionswere done with each DNA sample. A total of 70 clones were selected and combined for re-striction fragment length polymorphism (RFLP) analysis. Plasmids containing inserts wereextracted using an alkaline lysis miniprep standard method (Ausubel, Brent, Kingston,Moore, Seidman, Smith, and Strujl 1990). Plasmids were digested with EcoRI and RsaI(New EnglandBiolabs,Beverly,Massachusetts) usingconditionssuggestedby themanufac-turer. RFLP patterns were separated using 1.8% agarose gel electrophoresis and comparedfollowing ethidium bromide staining.

DNA Sequencing and Phylogenetic Analysis

One clone sequence from each unique RFLP group was ampli� ed using the primer pairs8F/1492R and puri� ed using a Wizard PCR Prep DNA puri� cation kit (Promega, Madison,Wisconsin). Automated DNA sequencing was done on a single strand using the 8F primerwith an Applied Biosystems Model 373A automated sequencer (Foster City, California).

DNA sequences were submitted to the CHECK CHIMERA program of the Riboso-mal Data Base Project (RDP) at Michigan State University to detect possible chimericartifacts (Maidak et al. 1999) using the SIMILARITY RANK program of the RDP, ini-tially aligned against other closely related rRNA sequences obtained from the RDP us-ing Clustal W (Thompson, Higgins, and Gibson 1994), followed by manual adjustmentbased on conserved primary and secondary structure. Phylogeneticanalysis was carried outusing maximum likelihood, neighbor joining, and maximum parsimony as implementedby PAUP¤ (Swofford 1996). The model of evolution used for likelihood searches was de-termined using MODELTEST (Posada and Crandall 1998). To estimate reliability in theconstructed bacteria clades, 1000 parsimony bootstrap replicates were run on the dataset.


TABLE 1 Proportion of rRNA clones, phylogenetic af� liation, andGenBank Accession numbers obtained from Cesspool Cave microbialmats

RFLP Clones recovered GenBankGroup (%)a Phylogenetic af� liationb Accession No.

"-ProteobacteriaCC-4 30.0 Helicobacter group AF207530CC-9 17.1 Helicobacter group AF207534

° -ProteobacteriaCC-5 28.5 Thiothrix group AF207531CC-7 15.7 Thiothrix group AF207532CC-8 4.3 Bacteriodes-Cytophaga- AF207533

Flavobacterium phylum

aCalculated by dividing the number of group speci� c clones by the total clones(n D 70).

bAs described by the Ribosomal Database Project.

Nucleotide Sequence Accession Numbers

The 16S rRNA gene sequences representing the clone groups obtained from CesspoolCave have been submitted to GenBank and assigned accession numbers AF207530 throughAF207534 (Table 1).

Results

Microbial Mat Structure

Microbial mats were distributed throughout the cave, with the densest concentrations nearthe spring and resurgence areas. Microbial mats in the cave pools consisted of thin, whiteto tan � laments that formed web-like structures at the sediment surface (Figure 2). Mats onthe cave walls near the pools formed a black to dark green gelatinousooze. No macroscopicbio� lms were observed in association with gypsum crusts.

SEM examination of microbial mats revealed that nonbranching � lamentous microor-ganisms dominated the mat structure from the back pool, whereas rod-shaped cells werecommon in the gypsum crusts (Figure 3). Both � laments and rods were associated withpool sediments (Figure 3A) and only � laments were abundant in samples from mats thatwere suspended in the water column (Figure 3B). Filamentous cells ranged from 2 to 4 ¹min diameter, whereas rod-shaped cells were approximately 1 to 1.5 ¹m in diameter fromsediment mats. However, cells from the water column were slightly smaller in diameter,ranging between 1 and 3 ¹m. Filaments from both mats showed evidence of septation andhormogonia but not rosettes that are common to Thiothrix (Brigmon et al. 1994). Both shortand long rods were observed on gypsum from the cave walls (Figure 3C and 3D). Somelong, rod-shaped cells were up to 3-¹m long and had cell diameters of 0.5 ¹m (Figure 3C).Other rods, ranging from 1 to 2.5 ¹m in length, appeared as single cells and in bio� lms ongypsum crystal faces.

Microbial Mat Ecology

Estimated rates of primary productivity for the Cesspool Cave pool microbial communitywere 30.4 § 12.0 ng C mgdw¡1 h¡1. Heterotrophic productivity was estimated as 0.17 §


FIGURE 2 Detail of Cesspool Cave, Virginia, microbial mats (white threads) in back poolattached to sediment surface (dark intermediary areas). Pen is 14 cm in length, and materialnear the pen is organic detritus.

0.02 ng C mgdw¡1 h¡1. To obtain the fraction of chemoautotrophic primary productionprocessed by heterotrophic bacteria, the ratio of heterotrophic to autotrophic productionwas calculated. Because the ratio of heterotrophic to autotrophic productivity does not re-� ect growth ef� ciency, the ratio was multiplied by 2 (assuming a 50% growth ef� ciency)(Kirchman, Keil, Simon, and Welschmeyer 1993). Using this method, the percent of au-totrophic productivity processed by heterotrophic metabolism was 1.3%. The carbon andnitrogen content of the mats, as an indication of microbial mat nutritional quality, had lowvalues of carbon and nitrogen, resulting in an average C:N ratio for the pool mats of 13.5,whereas the wall mats had a higher C:N ratio of 16.06.

16S rRNA Community Diversity

RFLP analysis of 70 clones indicated 7 different patterns, designated “clone group 4”through “clone group 10” (CC4 through CC10) (Table 1). Of the 7 groups, CC4 and CC5each represented approximately 30% of the clones, whereas CC6 through CC10 shared theremaining 40%. The CHECK CHIMERA program indicated that CC6 and CC10 were ofprobable chimeric origin and they were subsequently discarded from any further analysis.

The tree resulting from maximum parsimony analysis is shown in Figure 4. Maximum-likelihood and neighbor-joining analyses resulted in congruent trees with only minorchangesin branch length(datanot shown). Bothclones,CC5 andCC7, are ° -Proteobacteria,most closely related to Thiothrix ramosa and Thiothrix nivea. The clones CC4 and CC9grouped within the "-Proteobacteria, which includes spirochetes such as Geospirillum,Helicobacter, and Thiomicrospira, as well as Thiovulum. The most similar sequences to CC9were the two environmental clones JN5bf (Pederson, unpublisheddata) and G15, describedfrom groundwater in crystalline bedrock from Africa (Pedersen, Arlinger, Hallbeck, and


FIGURE 3 SEM photomicrographof microbial mats from CesspoolCave. A: Filamentousbacteria and sediment from pool.Scale bar is 10 ¹m. B: Mats from pool that were suspendedin the water column and grew in clumpsof web-like structures. Scale bar is 10 ¹m. C: Singlecells on gypsum crystal faces from gypsum on cave wall. Scale bar is 5 ¹m. Inset showsclose-up of a “long” rod-shaped bacterium in comparison to the rod in the main image.Scale bar is 1 ¹m. D: Bio� lm of short rods on gypsum substrate from cave wall. Scale baris 5 ¹m.

Petterson 1996). Clone CC8 clustered within the Bacteriodes–Cytophaga–Flavobacteriumphylum and was most closely related to environmental clone WCHB1-32, a phenol-degradingbacterial strain, and “Anaero�exus maritumus” (Woese, 1993, unpublisheddata).

Pure Culture Characterization

Liquid enrichment cultures of pool microbial mats in MS medium primarily containedshortand long rods, with lesser amounts of � lamentous- and coccid-shaped cells. Filamentousbacteria made up less than 15% of any enrichment cultures. In S1 enrichment cultures, allof the cells were Gram-negative rods.

More than 20 strains of chemoautotrophic bacteria were isolated in pure culture frompool and wall mats. Ten of these cultures were tested for acid production on BG andCaCO3 plates: 8 of the strains produced acid on the BG plates, but only 2 could dissolve


FIGURE 4 Maximum parsimony tree of ¯ -, ° -, and "-Proteobacteria and Bacteriodes-Cytophaga-Flavobacterium phylum rooted with Thermus aquaticus. Sequences isolatedfrom Cesspool Cave are indicated in bold. Numbers under the branches indicate per-cent bootstrap values for branches that were greater than 50%. Refer to Table 2 forCesspool Cave clone accession numbers. GenBank accession number for other sequencesare: Zoogloea, D84574; N. nitrosa, Z46986; T. thioparus, M79426; T. thermosulfatus(syn. Thiomonas thermosulfata), U27839; T. (syn. Thiomonas) perometabolis, M79421;L. disophora, L33974; B. cepacia, X87275; T. ramosa, U32940; T. nivea, U32940;M. luteus, X72772; M. pelagicum, X72775; L. mucor, X87277; symbiont of T. � exuosa,L01575; symbiont of S. velum, M90415; E. shaposhnikovii, X93480; B. alba, AF110274;Geospirillium, U85965; Clone G15, X91187; Clone JN5bf, Z69270; H. pullorum, L36144;symbiont of R. exoculata, U29081; Pele’s Vent clone PVB 12, U15104; Pele’s Vent clonePVB 63, U15102;T. denitri�cans, L40808;Thiovulumsp., M92324;cloneSB5, AF029041;envWCHB1 32, AF050543; “A. maritimus,” unpublished; strain Phenol-4, AF121885;C. fermentans, M58768; Clone 22a, X89282; T. aquaticus, L09660.


TABLE 2 Summary of strains isolated from Cesspool Cave following screening onbromocresol green (BG) plates for acid-producing colonies

Sample Strain Colony Morphology Enrichment Dissolvelocation no. description and length media and pH CaCO3?

Sediment–Back pool 7 Round, white Rod, 1.2 ¹m MS, 6 NoSediment–Back pool 10 Round, clear Rod, 0.8 ¹m S2, 6 NoSediment–Back pool 18 Round, clear Rod, 0.8 ¹m MS, 6 YesSediment–Back pool 20 Round, clear Rod, 1.0 ¹m MS, 6 YesSediment–Front pool 1 Round, brown Rod, 1.5 ¹m MS, 4 NoResurgence 1A Flat, white Rod, 1.5 ¹m MS, 4 NoWall mat 1A Flat, brown Rod, 1.3 ¹m S1, 6 NoWall mat 1B Flat, brown Rod, 0.9 ¹m S1, 6 No

CaCO3 (Table 2). The 2 strains that cleared CaCO3, Cesspool strains 18 and 20, wereoriginally isolated on MS media, but could also grow on S1 medium. Phylogenetic analysisof these strains placed them within a clade of ¯ -Proteobacteria, most closely related to thechemoautotrophic sulfur-oxidizing bacteria Thiobacillus perometabolis and Thiobacillusthermosulfatus (Figure 4).

Discussion

Ecology and Chemoautotrophy in Sul� dic Caves

Caves are distinctive habitats associated with complete darkness, relatively constant atmo-spheric and water temperatures, and low concentrations of organic matter. Photosyntheticmaterial derived from surface input serves as the source of carbon that energeticallysupportsmost cave ecosystems. Researchers have proposed that the role of microorganisms in cavesis to serve as a food source for higher trophic levels (Dickson 1979); however, typically themicroorganisms cannot provide adequate energy to support a large, diverse ecosystem. Incontrast, the work of Sarbu et al. (1996) and Porter (1999) in Movile Cave, Romania, andby Sarbu, Galdenzi, Menichetti, and Gentile (2000) in the Frasassi Caves, Italy, suggestthat chemoautotrophic, sulfur-based microbial communities can generate enough energyas primary producers to sustain complex cave ecosystems. These caves receive little or nosurface-derived organic material, but instead they are dominated geochemically by reducedsulfur compounds in the cave waters.

Cesspool Cave appears to be dominated by chemoautotrophic bacterial activity, butthe rates of autotrophic productivity are low in comparison to other sul� dic karst microbialcommunities that have been studied (Porter 1999). Data collected by Mattison et al. (1998)from submarine caves with active sulfur-oxidizing microbial communities, however, hadsimilar overall autotrophic productivity rates to Cesspool Cave.

Only 1.3% of the autotrophic productivity in Cesspool Cave is being used by het-erotrophiccomponentsof the microbialcommunity.In comparison,valuesof bacterioplank-ton utilization of phytoplankton production in open ocean systems range from20–40% (Kirchman et al. 1993). The low percentageof heterotrophiccyclingof autotrophicproductivitysuggests that the majority of the organic carbon produced in the CesspoolCavecommunity is not being cycled through a detrital loop (Allan 1995). Furthermore, the nutri-tionalqualityof a food source for higher trophic levelshas been correlatedwith highnitrogencontent (Pandian and Marian 1986) and low C:N ratios (McMahon 1975). In Movile Cave


(Sarbu et al. 1996) and the Frasassi Caves (Sarbu et al. 2000; Vlasceanu et al. 2000), nutri-tional quality correlates positively with numbers of grazers and other higher trophic levelmacroinvertebrates that consume chemoautotrophic microbial mats. In contrast, the lownitrogen value and high C:N ratio indicate that the microbial mats in Cesspool Cave areprobably not utilized by higher organisms, which is not unexpected because there has beenno evidenceof grazers in the cave. A more thoroughcensus of macroinvertebrates is needed,however. Therefore, the majority of the chemoautotrophicproductivity in Cesspool Cave iseither lost from the system via stream transport, or could be processed heterotrophically inan unsampled, unknown portion of the system.

Cesspool Cave Community Composition

The 16S rDNA molecular analyses and laboratory culturing results from this study demon-strate that there is more microbial diversity in the Cesspool Cave system than previous worksuggested; however, both studies resulted in different views of community composition.Although culturing was useful for understanding metabolic requirements and viability ofsome microorganisms, it also introduced a selective bias toward those microorganismsthat grew fast and could be cultured in the laboratory (McDougald, Rice, Weichart, andKjelleberg 1998). To circumvent the negative aspects of culturing, a molecular analysis ofthe community was also done. Nucleic acid extraction and PCR-ampli� cation bias, how-ever, can also misconstrue microbial community structure (Reysenbach, Giver, Wickham,and Pace 1992; Siering 1998; Suzuki and Giovannoni 1996). Understanding the physio-logical basis behind geomicrobiological processes would be virtually impossible withoutculture studies, yet little would be known about the community structure of the most activegeomicrobiologicalhabitats without molecular analyses (Siering 1998).

Earlier microbiologicalwork conducted in Cesspool Cave focused on only three typesof sulfur-oxidizingbacteria,Beggiatoa,Thiothrix, and Achromatium, from the water columnand bio� lms on rock surfaces (Hubbard et al. 1990). CulturingThiothrixor other � lamentoussulfur-oxidizersis dif� cult (Brigmonet al. 1994;Lackey,Lackey,and Morgan1965), and it isnot surprising that these bacteriawere not isolated from CesspoolCave samples even thoughliquid enrichment cultures did contain some � lamentous cells. Hubbard et al. (1986, 1990)did not report any rod-shaped bacteria such as Thiobacillus from their microscopy study,although the only strains of bacteria isolated in the current work were Gram-negative, rod-shaped, chemoautotrophic sulfur-oxidizers. The lack of thiobacilli clones in the 16S clonelibrary, however, suggests that theymay be a minorcomponentof the community.Additionalapproaches, such as � uorescent in situ hybridization,are required to determine the distribu-tion of thiobacilli in these microbial mats consisting of predominately � lamentous bacteria.

Shallow water depths in Cesspool Cave promote the formation of an almost continuouslink between the sediment and the water surface by microbial mats. The SEM data suggestthat there are very little differences between the sediment mats and water column mats,other than the presence of mineral matter in the sediment. Hubbard et al. (1990) suggestthat Thiothrix occupies the sediment habitat and Beggiatoa forms the water column mats.Yet, the 16S rRNA molecular results of this study suggest that only Thiothrix is present.Thiothrixhas also been found in a numberof sul� dic springsand underwater, freshwater, andanchihaline caves in Florida (Brigmon et al. 1994; Lackey et al. 1965), and was identi� edby Angert et al. (1998) from the Sulphur River of Parker Cave. In contrast, both Thiothrixand Beggiatoawere recognizedmicroscopically from the submarine caves at Cape Palinuro(Mattison et al. 1998).

The identi� cation of microbes related to Helicobacter was surprising, as these bacteriaare microaerophilic, spiral-shaped, with some species causing gastritis and peptic ulcers


in humans (Gibson, Ferrus, Woodward, Xerry, and Owen 1999). There is no evidence thatSweet Springs valley receives water contaminated with human or animal wastes, and thename of the cave is only to reference the high concentrationsof hydrogen sul� de. Pedersen(1996) suggest that environmental clones associated with the Helicobacter subgroup maybe af� liated with the genus Thiovulum, an obligatechemoautotrophic,sulfur-oxidizingbac-terium from sul� dic groundwater habitats that deposits sulfur intracellularly (Stahl, Lane,Olsen, Heller, Schmidt, and Pace 1987). Cells are motile, can reach 25 ¹m in length, andgrow at sul� de and oxygen interfaces as veil- and web-like mats presumably due to apolysaccharide matrix (Smith and Strohl 1991). Thiovulum is also one of the microorgan-isms forming thick, white mats near hydrothermal vents (Moyer et al. 1995). Althoughboth Helicobacter and Thiovulum have single cells that are distinct morphologically, theseshapes were not observed through SEM examination or microscopy of laboratory enrich-ment cultures. Based on the fact that there is an abundance of reduced sulfur compounds,the occurrence of Thiovulum instead of Helicobacter is likely in Cesspool Cave; however,additional work needs to be done to resolve the identity of the CC4 and CC9 clones, es-pecially because CC4 was found to be one of the dominant clones in the 16S clone library(Table 1).

In addition to the obvious presence of � lamentous bacteria in sul� dic caves, thiobacilliare the most commonly identi� ed microorganisms from other systems, including MovileCave (Vlasceanu, Popa, and Kinkle 1997), Cupp-Coutunn Cave, Turkmenistan (Maltsev,Korshynov, and Semikolennykh 1997), Parker Cave (Angert et al. 1998), Cueva de VillaLuz, Mexico (Hose et al. 2000), and the Frasassi Caves (Vlasceanu et al. 2000). Mostcave thiobacilliare associated with extremely acidic conditions, such as the wall bio� lms inFrasassi Caves with a pH of 1 or less. Althoughno thiobacilliwere detected in our molecularanalysis of Cesspool Cave mats, we isolated several chemoautotrophic, sulfur-oxidizingstrains, including two related to T. perometabolis (Figure 4). Moreira and Amils (1997)recently suggestedthat the¯-Proteobacteriasulfur-oxidizerssuch as T. perometabolisand T.thermosulfatusbe transferred to the new genus Thiomonas due to their distinct mixotrophicmetabolismcompared to members of the genus Thiobacillus in the ° -Proteobacteriagroup.

The placement of clone CC8 in the Bacteriodes-Cytophaga-Flavobacterium phylumindicates there are bacteria present in the Cesspool Cave community that are not directly in-volvedwith cyclingof sulfur compounds.Members of this phylum,which is deeply rooted inthe domain Bacteria, have diverse metabolic capabilitiesand are commonly associatedwiththe degradation of complex biomolecules. Similar to clone CC8, a clone from Parker Cavewas also related to the environmental clone WCHB1-32 (Angert et al. 1998). The environ-mental cloneWCHB1-32 was described from a hydrocarbon-and chlorinated-contaminatedaquifer (Dojka, Hugenholtz, Haack, and Pace 1998), and the environmental clone Phenol-4was found in a sul� dogenic 2-bromophenol dehalogenatingand phenol-degradingcommu-nity (Knight, Kerkhof, and Haggblom 1999). The low percentage of CC8 clones and lowvalues of heterotrophic productivity, however, suggest that these microorganisms are notabundant in the Cesspool Cave mat community.

Geomicrobiological Impact of Sulfur-Oxidizing Bacteria

Because caves serve as unique access points into the subsurface, active sul� dic cave sys-tems such as Cesspool Cave are excellent models for studying the interactions of micro-bial ecology and geology. Palmer (1991) estimates that 10% of the world’s caves haveformed through sulfuric acid dissolution.Hydrogen sul� de can be oxidized through abioticautoxidation or by microorganisms, resulting in the production of sulfuric acid that cor-rodes calcium carbonate, and causes subsequent gypsum precipitation.Although thiobacilli


appear to form a small component of the microbial community in Cesspool Cave, they havethe potential to contribute signi� cantly to cave enlargement.

Substrate dissolution can be bene� cial for microorganisms due to the release of nutri-ents such as nitrogen and phosphorus in oligotrophic habitats (Rogers, Bennett, and Choi1998), but rock dissolution can also be detrimental, in the case of carbonates because ofpH � uctuations, and other rocks due to release of high concentrations of toxic compounds,including aluminum or trace metals (Rogers et al. 1999). Microbially enhanced corrosionof glasses and silicate rocks and the effects of essential nutrients and toxic elements on mi-crobial colonization are recent areas of investigation (Bennett, Hiebert, and Rogers 2000;Drewllo and Weissmann 1997), but the effects of weathering carbonates on microbial pop-ulations remains speculative. It is anticipated, however, that carbonate rock compositions,particularly accessory minerals forming the rock matrix, may also directly impact the dis-tribution of microorganisms, and perhaps this might explain colonizationpatterns in caves.Additionally, competition for essential nutrients and metabolic substrates may also play arole in governing the distribution of microorganisms in a speci� c habitat (Kuenen and Bos1989).

Of the possible effects of rock dissolution,the buffering capacityof the rock is assumedto have the greatest potential in� uence on microbial colonization in sul� dic caves. Acid-generatingbacteria growing in proximity to dissolvingcarbonatemust tolerate or overcomepH � uctuations if they are going to survive. Only two of the cultured strains from CesspoolCave, isolated from back pool mats, corroded CaCO3, even though other strains generatedacid on BG plates (Table 2). The high buffering capacity encountered on the CaCO3 platesmay have inhibited the growth of pH-sensitive, acidophilic bacteria.

In the Frasassi Caves and Cueva de Villa Luz, cave-wall bio� lms consisting of extremeacidophiles grow on thick crusts of gypsum and elemental sulfur that have presumablyformed as a result of microbial activities (Hose et al. 2000; Vlasceanu et al. 2000). Thesebacteria are able to maintain highly acidic conditions and to avoid extreme pH changesby growing separately from the carbonate host rock. Microbially generated acid formed inthe bio� lms diffuses through the gypsum to the carbonate surface or drips from the tips ofthe microbial bio� lms onto exposed carbonate surfaces, thereby causing rock dissolution.Vlasceanu et al. (2000) also suggest that large portions of the crusts may detach from thewalls and expose fresh carbonate. There are minor accumulations of gypsum on CesspoolCave walls, and although some microorganisms were observed using SEM, the cave-wallbio� lms are not as extensive nor do they generate measurable quantities of acid like othercave-wall bio� lms in the Frasassi Caves or Cueva de Villa Luz. Therefore, it is unlikelythat such subaerial speleogenetic processes are dominating Cesspool Cave formation, andcarbonate dissolution is probably occurring at a greater extent within the cave pools.

In conclusion, the Cesspool Cave sulfur-oxidizing bacteria that grow in associationwith sediments may do so because the sediment confers bene� cial conditions and, muchlike gypsum crusts on the cave walls, may also allow bacteria to maintain acidic conditionslocallywhile acid can readily diffuse into the water and corrode nearby carbonate surround-ings. Consequently, chemoautotrophic processes dominate the microbial mat communityfrom Cesspool Cave, but these bacteria are not productive enough to support higher trophiclevels ecologically. Nevertheless, carbonate rock dissolution due to the microbial activityof sulfur-oxidizing and acid-producing bacteria has tremendous geological implications.Sulfuric acid dissolution has been proposed for the formation of several karst systems,including the caves of the Guadalupe Mountains of Texas and New Mexico (e.g., CarlsbadCavern and Lechuguilla Cave; Hill 1996), and karsti� ed carbonate oil� elds having signif-icant cavernous porosity, such as the Lisburne � eld in Prudhoe Bay, Alaska (Hill 1995;Jameson 1994). If active sul� dic karst systems are characterized by sulfuric acid reactions


derived from both abiotic and biotic processes, it can be assumed that these ancient sulfuricacid karst systems had the same processes occurring.

References

Allan DJ. 1995. Stream ecology: Structure and function of running waters. New York: Chapman andHall. 388 p.

Angert ER, Northup DE, Reysenbach AL, Peek AS, Geobel BM, Pace NR. 1998. Molecular phylo-genetic analysis of a bacterial community in Sulphur River, Parker Cave, Kentucky.Am Mineral83:1583–1592.

Ausubel F, Brent R, Kingston R, Moore D, Seidman J, Smith H, Strujl K, editors. 1990. Currentprotocolsin molecularbiology.New York:Greene PublishingAssociatesandWiley-Interscience,vol 1, suppl. 15. p 1.6.1–1.6.2.

Bennett PC, Hiebert FK, Rogers JR. 2000. Microbial control of mineral-groundwater equilibria:macroscale to microscale. Hydrogeol J 8:47–62.

Brierley JA. 1966. Contributionof chemoautotrophicbacteria to the acid thermal waters of the GeyserSprings Group in Yellowstone National Park. PhD dissertation. Bozeman, MT: Montana StateUniversity.

Brigmon R, Martin H, Morris T, Bitton G, Zam S. 1994. Biogeochemical ecology of Thiothrix spp.in underwater limestone caves. Geomicrobiol J 12:141–159.

Brock TD, Mosser JL. 1975. Rate of sulfuric-acidproductionin YellowstoneNational Park. Geol SocAm Bull 86:194–198.

Deming J, Baross J. 1993. Deep-sea smokers: windows to a subsurface biosphere? GeochimCosmochim Acta 57:3219–3230.

Dickson G. 1979. The importance of cave mud sediments in food preferences, growth and mortalityof the troglobitic invertebrates.Natl Speleol Soc Bulletin 37:89–93.

Dojka M, Hugenholtz P, Haack S, Pace NR. 1998. Microbial diversity in a hydrocarbon- andchlorinated-solvent-contaminated aquifer undergoing intrinsic bioremediation. Appl EnvironMicrobiol 64:3869–3877.

Drewello R, Weissmann R. 1997. Microbially in� uenced corrosionof glass. Appl MicrobiolBiotech-nol 47:337–346.

Edwards KJ, Bond PL, GihringTM, Ban� eld JF. 2000.An archaeal iron-oxidizingextreme acidophileimportant in acid mine drainage. Science 287:1796–1799.

Gibson JR, Ferrus MA, Woodward D, Xerry J, Owen RJ. 1999. Genetic diversity in Helicobacterpul-lorum from human and poultry sources identi� ed by an ampli� ed fragment lengthpolymorphismtechnique and pulsed-� led gel electrophoresis.J Appl Microbiol 87:602–610.

Harrison AP. 1984. The acidophilic thiobacilli and other acidophilic bacteria that share their habitat.Annu Rev Microbiol 38:265–292.

Hill CA. 1995. H2S-related porosity and sulfuric acid oil-� eld karst. In: Budd D, Saller A, HarrisP, editors. Unconformities and porosity in carbonate strata. Am Assoc Petroleum Geol Memoir63:301–306.

Hill CA. 1996. Geology of the Delaware Basin, Guadalupe, Apache, and Glass Mountains, NewMexico and West Texas. Permian Basin Section (SEPM) Pub. No. 96-39 480p.

Hose LD, Palmer AN, Palmer, MV, Northup DE, Boston, PJ, DuChene, HR. 2000. Microbiologyandgeochemistry in a hydrogen-sulphiderich karst environment.Chem Geol 169:399–423.

Hubbard D, Herman J, Bell P. 1986. The role of sul� de oxidation in the genesis of Cesspool Cave,Virginia, USA. Proc Int Congr Speleol, Barcelona, Spain 9:255–257.

Hubbard D, Herman J, Bell P. 1990. Speleogenesis in a travertine scarp: observations of sul� deoxidation in the subsurface.In: Herman J, Hubbard D, editors.Travertine-marl:Stream depositsin Virginia. Pub 101. Charlottesville,VA. Virginia Division of Mineral Resources. p 177–184.

Jackson C, Harper J, Willoughby D, Roden E, Churchill P. 1997. A simple, ef� cient method for theseparationof humic substancesand DNA from environmentalsamples. Appl Environ Microbiol63:4993–4995.

Jameson J. 1994. Models of porosity formation and their impact on reservoir description in theLisburne � eld, Prudhoe Bay, Alaska. Am Assoc Pet Geol Bull 78:1651–1678.


Johnson D. 1998. Biodiversity and ecology of acidophilic microorganisms. FEMS Microbiol Ecol27:307–317.

JørgensenNOG. 1992.Incorporationof [3H]leucineand [3H]valineinto proteinof freshwaterbacteria:uptake kinetics and intracellular isotope dilution. Appl Environ Microbiol 58:3638–3646.

Kirchman DL. 1993. Leucine incorporation as a measure of biomass production by heterotrophicbacteria. In: Kemp PF, Sherr BF, Sherr EB, Cole JJ, editors. Handbook of methods in aquaticmicrobial ecology. Lewis Publishers. p 509–512.

Kirchman DL, Keil RG, Simon M, WelschmeyerNA. 1993. Biomass and productionof heterotrophicbacterioplanktonin the oceanic subarctic Paci� c. Deep-Sea Res I 40:967–988.

Knight VK, Kerkhof LJ, and Haggblom, MM. 1999. Community analyses of sul� dogenic 2-bromophenol-dehalogenating and phenol-degradingmicrobial consortia. FEMS MicrobiolEcol29:137–147.

Kuenen JG, Bos P. 1989. Habitats and ecological niches of chemolitho(auto)trophic bacteria. In: HGSchlegel,BBowien,editors.Autotrophicbacteria.Madison,WI:ScienceTechPublishers.p53–80.

KuenenJ,RobertsonL,TuovinenO.1992.The generaThiobacillus,Thiomicrospira, andThiosphaera.In: Balows A, Truper H, Dworkin M, Harder W, Schleifer K, editors. The prokaryotes. Vol. 3.New York: Springer-Verlag. p 2638–2657.

Lackey JB, Lackey EW, Morgan GB. 1965. Taxonomy and ecology of the sulfur bacteria. FloridaEngineering and Industrial Experiment Station, College of Engineering, University of Florida,Gainesville, Bulletin Series No. 119. 19:23p.

Lane D, Pace B, Olsen G, Stahl D, Sogin M, Pace N. 1985. Rapid determination of 16S ribosomalsequences for phylogenetic analyses. Proc Natl Acad Sci USA 82:6955–6959.

Lowe D, Gunn J. 1995.The role of strongacid in speleo-inceptionandsubsequentcaverndevelopment.In: Barany-Kevei I, Mucsi L, editors. Special issue Acta Geographica (Szeged) 34:33–60.

Maidak B, Cole J, Parker C, Garrity G, Larsen N, Li B, Lilburn T, McCaugheyM, Olsen G, OverbeekR, Pramanik S, Schmidt T, Tiedje J, Woese C. 1999. A new version of the RDP (RibosomalDatabase Project). Nucl Acid Res 27:171–173.

Maltsev V, KorshynovV, SemikolennykhA. 1997.Cave chemolithotrophicsoils. In: JeanninP, editor.12th Int Congr Speleol, La-Chaux-de-Fonds,Switzerland. p 29–32.

Mattison R, AbbiatiM, Dando P, FitzsimonsM, Pratt S, Southward A, Southward E. 1998. Chemoau-totrophic microbial mats in submarine caves with hydrothermal sulphidic springs at CapePalinuro, Italy. Microb Ecol 35:58–71.

McDougald D, Rice S, Weichart D, Kjelleberg S. 1998. Nonculturablility:adaptationor debilitation?FEMS Microbiol Ecol 25:1–9.

McMahon RF. 1975. Growth, reproductionand bioenergeticvariation in three natural populations ofa freshwater limpet Laevapex fusus (CB Adams). Proc Malacol Soc London 41:331–342.

Milde K, Sand W, Wolff W, Bock E. 1983. Thiobacilli of the concrete corroded walls of the Hamburgsewer system. J Gen Microbiol 129:1327–1333.

Moreira D, Amils R. 1997. Phylogeny of Thiobacillus cuprinus and other mixotrophic thiobacilli:proposal for Thiomonas gen. nov. Int J Syst Bacteriol 47:522–528.

Moyer C, Dobbs F, Karl D. 1995. Phylogeneticdiversity of the bacterial community from a microbialmat at an active, hydrothermal vent system, Loihi Seamount, Hawaii. Appl Environ Microbiol61:1555–1662.

Nation JL. 1983. A new method using hexamethyldisilazanefor preparation of insect soft tissues forscanning electron microscopy. Stain Technol 58:347–351.

Palmer AN. 1991. Origin and morphology of limestone caves. Geol Soc Am Bull 103:1–21.Pandian TJ, Marian MP. 1986. An indirect procedure for the estimation of assimilation ef� ciency of

aquatic insects. Freshwater Biol 16:93–98.Parker CD, Jackson D. 1965. The microbial � ora of concrete surfaces. Hydrogen sul� de corrosion of

concrete sewers: Melborne and Metropolitan Board of Works Tech Paper No. A8, v 6, p 1–29.Pedersen K. 1996. Investigations of subterranean bacteria in deep crystalline bedrock and their im-

portance for the disposal of nuclear waste. Can J Microbiol 42:382–391.Pedersen K, Arlinger J, Hallbeck L, Pettersson C. 1996. Diversity and distribution of subterranean

bacteria in groundwater at Oklo in Gabon, Africa, as detected by 16s rRNA gene sequencing.Mol Ecol 5:427–436.


Porter M. 1999. Ecosystem energetics of sul� dic karst. Master’s thesis. Cincinnati, OH: Universityof Cincinnati.

Posada D, Crandell KA. 1998. MODELTEST: testing the model of DNA substitution.Bioinformatics14:817–818.

Reysenbach AL, Giver LJ, Wickham GS, Pace NR. 1992. Differential ampli� cation of rRNA genesby polymerase chain reaction. Appl Environ Microbiol 58:3417–3418.

Rogers JR, Bennett PC, Choi WJ. 1998. Feldspars as a source of nutrients for microorganisms. AmMineral 83:1532–1540.

Rogers JR, Bennett PC, Hiebert FK. 1999. Patterns of microbial colonization on silicates. In:Morganwalp DW, Buxton HT, editors.Water Resources InvestigationsReport 99-4018C, vol. 3.U.S. Geol Surv Toxic Substances Hydrology Program. p 237–242.

Sarbu S. 1996. A chemoautotrophicallybased groundwater ecosystem. PhD dissertation.Cincinnati,OH: University of Cincinnati.

Sarbu S, Kane T, Kinkle B. 1996. A chemoautotrophically based groundwater ecosystem. Science272:1953–1955.

Sarbu S, Galdenzi S, Menichetti M, Gentile G. 2000. Geology and biology of Grotte di Frasassi(Frasassi Caves) in Central Italy, an ecological multi-disciplinary study of a hypogenic under-ground karst system. In : Wilkens H, Culver D, Humphreys W, editors.Ecosystems of the world,vol. 30: Subterranean Ecosystems. Oxford, UK: Elsevier Science. p 361–381.

Sarbu S, Popa R. 1992. A unique chemoautotrophically based cave ecosystem. In: Camacho AI,editor. The natural history of biospeleology.Madrid: Monogra�as Museo Nacional de CienciasNaturales. p 637–666.

Siering P. 1998. The double helix meets the crystal lattice: the power and pitfall of nucleic acidapproaches for biomineralogicalinvestigations.Am Mineral 83:1593–1607.

Smith D, Strohl W. 1991. Sulfur-oxidizing bacteria. In: Shively J, Barton L, editors. Variations inautotrophic life. London: Academic Press, 356p.

Smyk B, Drzal M. 1964. Research on the in� uence of microorganisms on the development of karstphenomena. Geographica Polonica 2:57–60.

Stahl DA, Lane DJ, Olsen GJ, Heller D, Schmidt T, Pace N. 1987. Phylogenetic analysis of certainsul� de-oxidizingand related morphologicallyconspicuousbacteriaby 5S ribosomal ribonucleicacid sequences. Int J Syst Bacteriol 37:116–122.

Suzuki MT, GiovannoniSJ. 1996. Bias casued by template annealing in the ampli� cation of mixturesof 16S rRNA genes by PCR. Appl Environ Microbiol 62:625–630.

Swofford P. 1996. PAUP¤: phylogenetic analysis using parsimony (and other methods), ver. 4.0.Sunderland, MA: Sinaeur Associates.

Thompson JD, Higgins DG, Gibson TJ. 1994. CLUSTAL W: improving the sensitivityof progressivemultiple sequence alignment through sequence weighting, position-speci�c gap penalties andweight matrix choice. Nucl Acid Res 22:388–392.

Vlasceanu L, Popa R, Kinkle B. 1997. Characterization of Thiobacillus thioparus LV43 and itsdistribution in a chemoautotrophicallybased groundwater ecosystem. Appl Environ Microbiol63:3123–3127.

VlasceanuL, Sarbu SM, Engel AS, Kinkle BK. 2000.Acidic cave-wallbio� lms located in the FrasassiGorge, Italy. Geomicrobiol J 17:125–139.

Wagner M, Schwartz W. 1967. GeomikrobiologischeUnterschungen. VIII. Uber das Verhalten vonBakterien auf der Ober� ache von Gesteinenund Mineralienund ihre Rolle bei der Verwitterung.A Allg Mikrobiol 7:33–52.

Wetzel RG, Likens GE. 1991. Limnological analyses, 2nd ed. New York: Springer-Verlag. 391p.Wilson WL, Morris TL. 1994. Cenote Verde: A meromictic karst pond, Quintana Roo, Mexico. In:

Sasowsky I, Palmer M, editors.Breakthroughsin karst geomicrobiologyand redoxgeochemistry.Karst Waters Institute, Special Publication. Vol. 1, p 77–79.

ZherebyatevaT, Lebedeva E, Karavaiko G. 1991. Microbiologicalcorrosion of concrete structures ofhydraulic facilities. Geomicrobiol J 9:119–127.

Zhou J, Bruns M, Tiedje J. 1996. DNA recovery from soils of diverse composition. Appl EnvironMicrobiol 62:316–322.

ecological assessment and geological significance of ...dzumenvis.nic.in/microbes and metals...

Documents