Geomicrobiology Journal, 21:169–182, 2004Copyright C© Taylor & Francis Inc.ISSN: 0149-0451 print / 1362-3087 onlineDOI: 10.1080/01490450490275848

Microbiological Comparison of Core and GroundwaterSamples Collected from a Fractured Basalt Aquifer withthat of Dialysis Chambers Incubated In Situ

R. Michael Lehman,1 Sean P. O’Connell,1 Amy Banta,2 James K. Fredrickson,3

Anna-Louise Reysenbach,2 Thomas L. Kieft,4 and Frederick S. Colwell1

1Biotechnology Department, Idaho National Engineering and Environmental Laboratory, Idaho Falls,Idaho, USA2Department of Environmental Biology, Portland State University, Portland, Oregon, USA3Department of Environmental Microbiology, Pacific Northwest National Laboratory, Richland,Washington, USA4Biology Department, New Mexico Institute of Mining and Technology, Socorro, New Mexico, USA

Microorganisms associated with basalt core were compared tothose suspended in groundwater pumped from the same well in theeastern Snake River Plain Aquifer (Idaho, USA). Two wells locatedat different distances from the source of a mixed-waste plume inthe fractured basalt aquifer were examined. In the well more distalfrom the plume source, an array of dialysis chambers filled witheither deionized water or crushed basalt was equilibrated to com-pare the microorganisms collected in this fashion with those fromcore and groundwater samples collected in a traditional mannerfrom the same well. The samples were characterized to determinethe total amount of biomass, presence of specific populations orphysiological groups, and potential community functions. Microor-ganisms and their activities were nearly undetectable in core andgroundwater collected from the well farthest from the plume sourceand substantially enriched in both core and groundwater from thewell closest to the plume source. In both wells, differences (statisti-cally significant for some measures) were found between bacteria

Received 10 December 2002; accepted 3 September 2003.This research was funded by the U.S. Department of Energy, Office

of Environmental Management, Environmental Management ScienceProgram, by grant to FSC at the Idaho National Engineering and En-vironmental Laboratory (INEEL) operated at that time by LockheedMartin Idaho Technologies Co. under contract DE-AC07-94ID13223and subsequently operated by Bechtel BWXT, LLC under contract DE-AC07-99ID13727. Assistance in laboratory analyses or field samplingis acknowledged from the following persons: Matt Downing, MarkDelwiche, Brad Blackwelder, Travis McLing, Mark Wilson, Pete Pry-fogle, Bob Smith, Shu-mei Li, Jim McKinley, and Teresa Wilson. JayAnderson and Maribeth Watwood are acknowledged for critical reviewof the manuscript.

Address correspondence to R. Michael Lehman, USDA/ARS/Northern Grain Insects Research Laboratory, 2923 MedaryAve., Brookings, SD 57006, USA. E-mail: mlehman@ngirl.ars.usda.gov

associated with the cores and those suspended in the groundwa-ter. Significantly higher populations were found in the basalt- andwater-filled dialysis chambers incubated in the open well comparedwith core and groundwater samples, respectively. For a given pa-rameter, the variation among dialysis chambers incubated at differ-ent depths was much less than the high variation observed amongcore samples. Analyses on selected basalt- and water-filled dialy-sis chamber samples suggested that these two communities werecompositionally similar but exhibited different potential functions.Documented knowledge of cell physiological changes associatedwith attachment and potential differences between attached andunattached communities in aquifers indicate that careful consid-eration should be given to the type of sample media (i.e., core,groundwater, substrata incubated in a well) used to represent asubsurface environment.

Keywords aquifer, attached, free-living, groundwater, microbiology,subsurface, TCE

INTRODUCTIONAlthough groundwater and solid geologic media have been

shown to contain significant biomass and diversity (Pedersen2000), microbiological characterization of subsurface environ-ments has generally focused on samples of either groundwateror core, but rarely both. Based on the apparent predominanceof attached biomass observed in unconsolidated, sedimentaryaquifers, it is often concluded that core samples are most rep-resentative of the subsurface (Harvey et al. 1984; Ghiorse andWilson 1988; Pedersen and Ekendahl 1990; Hazen et al. 1991;Alfreider et al. 1997). In contrast, some studies have focusedlargely on groundwater samples (Gounot 1994). It seems prob-able that attached and free-living aquifer microbial communities

169

170 R. M. LEHMAN ET AL.

may be compositionally and functionally different, howeverthere is little recognition of this possibility and surprisingly littledata to support this conclusion. In the few studies that have ex-amined microbial community composition in depth-paired coreand groundwater samples from a single borehole, compositionaldifferences have been observed in unconsolidated sedimentaryaquifers (Kolbel-Boelke et al. 1988; Godsy et al. 1992; Bekinset al. 1999) and in a fractured rock aquifer (Lehman et al. 2001).A description of biomass distribution in fractured rock aquifersis confounded by the lack of equitable units of comparison.

Coring crystalline rock or sedimentary deposits to obtainuncontaminated samples for microbiological analysis is morecostly and technically challenging than drilling a well and pump-ing groundwater samples. Given these disadvantages, usinggroundwater samples for biodegradation feasibility studies andin situ bioremediation monitoring would be advantageous; how-ever, there is uncertainty about the applicability of those resultsto in situ processes (Thomas et al. 1987; Hirsch and Rades-Rohkohl 1988; Alfreider et al. 1997). Alternatively, it is difficultto justify the expense of coring if equal uncertainty exists withrespect to representation of aquifer microbiota by the core sam-ples. Some researchers have incubated substrata in wells or ingroundwater flowing from wells to interrogate the attached mi-crobiota while avoiding the cost and technical difficulties ofcoring (Hirsch and Rades-Rohkohl 1990; Holm et al. 1992;Pedersen and Ekendahl 1992; Pedersen et al. 1996; Alfreideret al. 1997). However, no detailed comparisons exist that supportthe accurate representation of communities attached to geologicsubstrata by those attached to incubated substrata.

The fact that groundwater invariably contains microorgan-isms and these microorganisms may be different from thoseobserved in corresponding core samples suggests an incompleteconceptual view of the biological component of subsurface en-vironments. Research findings have shown that changes in adiversity of metabolic traits are associated with a cell becom-ing attached to a surface (Van Loosdrecht et al. 1990; Marshall1992). Several authors have demonstrated the effects of cell at-tachment on the biodegradation rates of organic contaminants(Holm et al. 1992; Doong et al. 1997). Holm et al. (1992) statedthat the distinction of biodegradation potential between attachedand free-living biological compartments in aquifers is “largelyunexplored”. It has been shown that sorption of organic com-pounds to substrata influences their bioavailability and attach-ment of microorganisms to the same substratum is related totheir access to the sorbed compound (Guerin and Boyd 1997).Modeling of benzoate biodegradation in porous media basedon a meso-scale experimental model has implicated the parti-tioning of biodegrading populations as a factor in the observedtransport of the benzoate (Murphy et al. 1997). If populationsare partitioned unequally between attached and free-living statesand cell physiology alters upon attachment, then the two com-munities may possess substantially different functional roles.

In order to address differences in attached and free-livingaquifer communities and their potential roles in biogeochemical

transformations, we compared the microbiology of groundwa-ter and core from two boreholes in the fractured basalt of theeastern Snake River Plain Aquifer. The groundwater in one bore-hole was suboxic due to its proximity to the source of a mixed-waste plume that contains sewage and trichloroethylene (TCE).In the oxic groundwater of the second borehole more distal tothe plume source, dialysis chambers filled with either deion-ized water or crushed basalt previously cored from that wellwere equilibrated in the well. The different types of sampleswere compared by estimates of total biomass or cells, commu-nity composition evidenced by population abundances for dif-ferent physiological groups and 16S rDNA analysis of a limitednumber of samples, and community functional potential as ev-idenced by mineralization of C-14 labeled acetate and patternsof community sole carbon source utilization. The enumerationsof physiological groups reported to participate in TCE degra-dation under oxic and suboxic conditions revealed additionalinformation regarding the potential for biological reduction ofchlorinated hydrocarbon concentrations.

MATERIALS AND METHODS

Field SamplingSite Description. Samples from two wells, TAN-33 and

TAN-37, were used to compare the properties of attached andunattached microorganisms. The wells are located at the IdahoNational Engineering and Environmental Laboratory at the TestArea North facility (Figure 1). The wells penetrated the layeredbasalt flows and occasional thin sedimentary interbeds of theeastern Snake River Plain Aquifer to a total depth of 134 m(TAN-33) and 126 m (TAN-37) below land surface (bls). At thislocation the water table is approximately 63–65 m bls and meangroundwater flow velocities are on the order of 0.2 m day−1 (K.Sorenson, personal communication). The only sedimentary in-terbeds at this location are close to the depth of the water tableand at the very base of the wells. The upper interbed was cased-off and the encounter with the lower interbed indicated that totalwell depth was reached and coring was terminated. Therefore, allcore samples reported in this paper are basalt and all groundwatersamples were pumped from intervals dominated by basalt. Al-though groundwater is contained within the basalt matrix, move-ment of groundwater is largely through fractures and the rubblezones that characterize basalt flow interfaces. The groundwa-ter is calcium-sodium-bicarbonate based with high silica andgenerally saturated levels of dissolved oxygen flowing throughyoung, olivine basalt (Wood and Low 1986). Both wells are lo-cated downgradient from a former injection well used between1953 and 1972 for waste disposal of chlorinated hydrocarbons,sewage, and low-level radioactive liquid waste. The two wellsare influenced to different degrees by the mixed-waste plume asevidenced by their respective groundwater chemistries (Table 1).

Core Samples. Basalt cores were obtained from the satu-rated zone by reverse air-rotary coring with the addition of a

SUBSURFACE MICROBIAL SAMPLING 171

Figure 1. Location of field sampling site at the Idaho National Engineering and Environmental Laboratory Test Area Northfacility.

mist of disinfected (10 mg liter−1 sodium hypochlorite) ground-water added to the air to assist in bit cooling and removal of cut-tings. Control and assessment of microbial contamination intro-duced during coring were consistent with procedures reviewedby Fredrickson and Phelps (1997) and Griffin et al. (1997), in-cluding use of carboxylated, fluorescent microspheres (0.9 µmdiameter; Polysciences, Inc., Warrington, PA) (Russell et al.1992) and soluble perfluorocarbon tracers (PFT) (McKinley andColwell 1996) to assess drilling fluid intrusion into the cores.Cores were aseptically processed on-site under an O2-free, ar-

gon atmosphere (Fredrickson and Phelps 1997). Nineteen coreswere collected from TAN-33 between 70 and 134 m bls and 23cores from TAN-37 between 63 and 126 m bls for microbio-logical analyses. In the laboratory, basalt core fragments werecrushed further using a sterile mortar and pestle. The crushedbasalt was then blended with 0.1% (w/v) sodium pyrophosphate(Balkwill and Ghiorse 1985) and this slurry was used as an in-oculum for culture-based analyses. The inoculum included smallparticulate and colloidal basalt that did not settle immediatelyfollowing the termination of blending.

172 R. M. LEHMAN ET AL.

Table 1Characteristics of wellsa

Parameter Well TAN-33 Well TAN-37

Distance from waste injectionwell (m) 425 35

Dissolved oxygen (mg/l) 6–7 3–4pH (−log[H+]) 8 8Temperature (◦C) 12–13 12–13Total organic carbon (mg/l) <0.5 1Total chlorinated alkenes (mg/l) <1 1–5Conductivity (µS/cm) 600 7003H (µCi/l) 4 490Sr (picoCi/l) <0.2 150Orthophosphate (mg/l) <1.0 <1.0Nitrate (mg/l) 9 10Ammonium (mg/l) <0.1 0.5Sulfate (mg/l) 30 40Chloride (mg/l) 80 90Calcium (mg/l) 72,000 68,000Magnesium (mg/l) 18,000 21,000Potassium (mg/l) 3,300 4,800Sodium (mg/l) 25,000 40,000

aData are nominal values taken from Bukowski, J. 2000. Fiscal Year1999 Groundwater Monitoring Annual Report Test Area North, Oper-able Unit 1-07B, INEEL/EXT-99-01255, U.S. Department of Energy,January 2000.

Groundwater Samples. For TAN-33, five bulk groundwa-ter samples were collected with a pump placed at 84 m bls inthe open well following standard three-well-volume purging.For TAN-37, three depth intervals were isolated with a strad-dle packer (Shuter and Pemberton 1978) and groundwater wassampled from each of these three zones following purging (threevolumes). The midpoints of the 6-m long packed-off intervalswere 85.9 m, 95.7 m, and 113.4 m. Groundwater samples werecollected in sterile containers, placed on ice, and transported tothe laboratory for analysis.

Sampling with Dialysis Chambers. For TAN-33 only, sam-ples for microbiology were collected from between 94.5 m and114.5 m bls (a depth interval containing two highly conductiverubble zones and no sedimentary interbeds) using a string of 35-cm3 dialysis chambers deployed in a series of polyvinyl chloride(PVC) rods that were lowered into the uncased well (Figure 2).This sampler design has been used to investigate groundwatergeochemistry (Ronen et al. 1986) and in situ biodegradation oforganic contaminants (Shati et al. 1996). Sets of seven dialy-sis chambers spanning a 0.35-m interval were separated fromadjacent intervals by Viton baffles fit to the local well diam-eter by study of the corehole caliper log. Within each set ofseven chambers, three chambers for microbiology had endcapsfitted with 10-µm pore-size dialysis membranes; the results ofanalyses performed on these three chambers are reported in thispaper. Dissolved constituents equilibrate across the membrane

Figure 2. Photograph of the device containing the basalt- andwater-filled dialysis chambers that were incubated in TAN-33. Aseries of 160-cm long, 5.7-cm diameter PVC rods were coupledtogether and lowered downhole so that it spanned an intervalfrom 94.5 m and 114.5 m bls. Fifty-five sets of seven dialysischambers (35-cm3) each spanning 0.35-m intervals were sepa-rated from adjacent intervals by Viton baffles fit to the local welldiameter by study of the corehole caliper log.

by diffusion, while suspended particulates, including bacteria,can potentially move into the chambers by diffusion, advection,chemotaxis, or growth after adhering to the membrane surface.In each of the 0.35-m intervals, two of the three chambers des-ignated for microbiological studies were filled with autoclaved,crushed basalt (1.6–3.2 mm diameter) previously cored fromthe well; the third chamber was filled with deionized water. Af-ter eight months’ equilibration in the well, the array of dialysischambers was retrieved and the chambers were removed fromthe string and placed into sterile bags and onto ice as rapidlyas the string of samplers could be uncoupled and raised. Thecontents of the dialysis chambers were decanted into sterile cen-trifuge tubes in random order to reduce systematic error in thedata. Out of 55 total intervals, 32 intervals spanning the entire20-m length of the array were analyzed for select microbio-logical properties. The water in the dialysis chambers was used

SUBSURFACE MICROBIAL SAMPLING 173

directly in the microbiological assays; crushed basalt wasblended with 0.1% (w/v) sodium pyrophosphate and the su-pernatant containing suspended particulate and colloidal basalt(but no large particles) was used as an inoculum.

Microbiological AnalysesTotal Bacterial Cell Counts and Phospholipid Fatty Acid

(PLFA) Analysis. Aliquots of formaldehyde-fixed (2%) sub-samples were filtered under vacuum onto 0.2-µm pore-size,black polycarbonate membrane filters with cellulose-acetate sup-port filters. Total bacterial cells were enumerated by direct countsof 4, 6-diamidino-2-phenylindole (DAPI)-(10 µg ml−1, 30 min)or acridine orange-stained (0.01%, 3 min) cells on the filtersunder epifluorescent illumination using a Zeiss #2 or Zeiss #9filter set, respectively (Kepner and Pratt 1994). A minimum offive fields and 200 cells was counted or 20 fields when 200 cellswere not achieved. PLFA analyses were performed on rock andgroundwater samples using standardized methods by MicrobialInsights (Knoxville, TN) (White 1979). Biomass was estimatedfrom the quantity of ester-linked phospholipid fatty acids and astructural community profile was generated based on the relativeabundance of phospholipid classes (White 1979).

Enumeration, Isolation, and Identification of Culturable Aer-obic Chemoheterotrophs. The number of culturable aerobicheterotrophs in samples was determined by standard dilutionplate methods on R2A solid medium (Reasoner and Geldre-ich 1985) after serial dilution of samples in sterile phosphate-buffered saline (PBS; 1.18 g Na2HPO4, 0.223 g NaH2PO4·H2O,and 8.5 g NaCl per liter; pH 7.3). The number of colony-forming-units (CFUs) were determined by examining plates 14 days af-ter incubation at room temperature in the dark. Morphologicallydistinct isolates from each sample were identified and enumer-ated using colony size, color, consistency, edge and elevationas parameters. For TAN-33 dialysis chamber samples from the103- and 113-m depth intervals, morphologically-distinct iso-lates were subsequently streaked until pure, and characterizedby determining the profiles of fatty acid methyl esters (FAME)using the MIDI system (MIDI, Inc., Newark, DE) per manufac-turer instructions.

Enumeration of Aerobic [Potential] Cometabolic TCEDegrading- and H2-Oxidizing Bacteria. Enumeration ofmethanotrophs was performed by most-probable number (MPN)analysis of sample dilutions in nitrate mineral salts (NMS) liq-uid medium (Bowman et al. 1993) modified by the additionof 1 mM (NH4)2SO4 to provide an alternative nitrogen sourcewith complementary reduction of NaNO3 from 2 mM to 1 mMand use of 16% methane in the headspace. For propanotrophs,16% propane was added to the headspace instead of methane.For phenol oxidizers, the same NMS was used with the addi-tion of 570 mg liter−1 phenol and an air-filled headspace. Aer-obic hydrogen-oxidizing bacteria were enumerated in the sameNMS medium described above with the addition of 0.2 mgliter−1 NiSO4·6H2O and adjustment of the headspace to 50%

hydrogen. The pH of the above NMS media was 7.5. Ammonia-oxidizers were enumerated in mineral salts medium (0.5 g(NH4)4SO4, 0.2 g KH2PO4, 0.04 g CaCl2·2H2O, 0.04 g MgSO4·7H2O and 0.5 mg ferric citrate per liter, pH 8.3) with 0.5 mgliter−1 phenol red added as an indicator (Atlas 1993). MPNmatrices for each sample, and positive (spiked with target or-ganism) and negative (uninoculated, sterilized inoculum and noadded organic carbon) controls were performed in 50-ml glassserum vials with static incubation at 22◦C. After three months’incubation, turbid vials were scored positive and growth wasconfirmed by direct microscopic observation of bacterial cellsand disappearance of methane, propane, hydrogen, or phenolas measured by gas chromatography. For ammonia-oxidizingbacteria, vials exhibiting loss of phenol red coloration due toproduction of acidity were tested for the presence of nitrate us-ing an ion-selective electrode.

Dissimilatory Iron-Reducing Bacteria. The presence ofacetate-oxidizing dissimilatory-iron reducing bacteria was de-termined by triplicate enrichments of 1 g or 1 ml of samplein mineral salts medium (Boone et al. 1995) with 40 mM hy-drous ferric oxide, 40 mM sodium acetate, and a headspace ratioof 1:5 CO2:N2 at a pH of 7.8. Confirmation of iron reductionon darkened enrichments was by comparing concentrations of0.5 N HCl-extractable ferrous iron by ferrozine assay in theenrichments to that in negative controls (Lovley and Phillips1986).

Community-Level Physiological Profiling (CLPP). CLPPwas performed by inoculating groundwater and basalt extractsinto Biolog GN microplates, which test the respiration of 95 dif-ferent sole carbon sources by the mixed communities (Garlandand Mills 1991). Extracts were prepared from the crushed basaltblended with sodium pyrophosphate (0.1%, pH 7.4) by agitatingthe basalt slurries at 150 rpm on a platform shaker for 24 h andthen separating the cells from abiotic particulates using gravitysettling with particle flocculation using 0.25 g of an 8:5 salts mix-ture of MgCO3:CaCl2·2H2O per 100 ml extractant (Demezasand Bottomley 1986). Microplates inoculated with the extrac-tant were incubated at 22◦C in a humidified atmosphere in thedark, and absorbance at 590 nm (i.e., production of reduced tetra-zolium dye coupled to carbon source oxidation) was recordedevery 2 hours for 1 week using an automated microplate reader-stacker (Multiscan MCC 340 MKII, ICN Pharmaceuticals, CostaMesa, CA). Results were considered in two ways: (i) commu-nity metabolic richness, which is simply the sum of all posi-tive carbon source utilization tests; and, (ii) community carbonsource utilization patterns based on the continuous absorbancereadings for each of the 95 reactions. Community carbon sourceutilization patterns were analyzed by principal components anal-ysis (PCA) (Rosswall and Kvillner 1978). Using background-corrected microplate readings at equivalent average well colordevelopment (Garland 1996; Garland 1997; O’Connell et al.2000), PCA was performed on the correlation matrix of thevariables (R matrix) with no factor rotation. The scores for eachsample on principal components factors 1 and 2 were plotted in

174 R. M. LEHMAN ET AL.

Figure 3. Experimental scheme using Biolog GN microplates to determine effectiveness of extraction of viable cells from basaltparticles.

two-dimensional factor space to examine the relationship amongthe samples.

Evaluation of Basalt Extraction Protocol for CLPP. CLPPrequired an extraction procedure to generate data on viable, at-tached bacteria that can be contrasted with their free-living coun-terparts. In a separate experiment, the Biolog GN microplateswere used to evaluate the completeness of the extraction of vi-able cells from the basalt (Figure 3). Crushed basalt from threedifferent TAN-33 dialysis chambers taken from the top, middle,and bottom of the array was extracted as described previously,and the extract was inoculated into the GN microplates (set 1).In a second set of GN microplates (set 2), the extracted parti-cles of basalt that remained after the supernatant was used toinoculate set 1 were placed into each of the 96 microwells andthe remainder of each microwell volume was filled with PBS.In a third set of GN microplates (set 3), unextracted basalt parti-cles from the same three dialysis chambers were incubated withPBS in the microwells that contained substrates that had notbeen respired by the basalt extracts (indicated by results of set1). All plates were incubated as previously described, and thesole carbon source utilization was recorded visually after 7 days.

Radiorespirometry. Aerobic and anaerobic mineralizationof 14C-labeled acetate were measured for each sample in trip-licate sterile bottles by the method of Kieft et al. (1995). Re-action bottles for cores received 10 g sediment (wet weight),9.8 ml artificial groundwater (3.8 mg NaF, 0.2 mg KBr, 7.2mg KCl, 36.4 mg CaCl2·2H2O, 32.7 mg MgCl2·6H2O, 82.2 mgNa2SiO3·9H2O and 899.8 mg NaHCO3 per liter), and 0.2 ml

of 1-14C sodium acetate (0.4 µCi ml−1, specific activity 2 µCiµmole−1, New England Nuclear). For TAN-33 dialysis cham-bers containing crushed basalt 5 g of basalt was mixed with4.8 ml of the artificial groundwater and the same amount of la-beled acetate as the core samples. Evolved, labeled CO2 wascollected in alkaline traps that were replaced at predeterminedintervals and radioactivity of 14CO2 at each time point was de-termined by liquid scintillation counting (14C, β). Acetate min-eralization was expressed as the percent mineralization of theadded labeled substrate after 3 days’ incubation.

DNA Extraction, Amplification, DGGE Analysis, and Se-quencing for TAN-33 Dialysis Chamber Samples (Basalt- andWater-Filled) from Depths of 103 and 113 m. DNA was ex-tracted from basalt (1.2 g) or water (filter retaining cells from40 ml of water) using a modified sucrose lysis method (Gordonand Giovannoni 1996). DNA extracted from the samples werecompared by denaturing gradient gel electophoresis (DGGE)(Muyzer et al. 1993; Rolleke et al. 1996). An approximately 180base fragment of the bacterial 16S rRNA gene was amplified byPCR from the DNA samples using the universally-conservedprimer (E.coli numbering) 519R and a bacterial-specific GC-clamp primer 338F-GC in a PCR mixture (50 µl) containing5 µl of 10X AmpliTaq PCR buffer (perkin-Elmer), 10 µmoldNTPs, 15 pmol of each primer, 0.05% IGEPAL (Sigma), and1.25 U of Amplitaq-LD (Perkin-Elmer). The reactions were in-cubated in a thermal cycler for 35 cycles under the followingconditions: 94◦C for 30 seconds, 50◦C for 30 seconds, and72◦C for 30 seconds with an initial denaturation at 94◦C for

SUBSURFACE MICROBIAL SAMPLING 175

3 minutes and final incubation at 72◦C for 10 minutes. Ampli-fied products (18 µl) were sorted on a denaturing gradient (20%to 60% urea/formamide) acrylamide (6%) gel for 2.5 hours at180 V and 60◦C in 1X TAE buffer (0.04 M Tris, 0.02 M ac-etate, and 1.0 mM EDTA). Gels were stained for 20 minutesin SYBR Green (1:20,000 dilution) and destained for 10 min-utes (Vetriani et al. 1999). Visible bands were reamplified, pu-rified, and sequenced. Bands were touched with a sterile pipettip and placed into 20 µl sterile 10 mM Tris for 5 minutes atroom temperature. Two to 8 µl of this solution was used ina PCR amplification using 20 pmol of the primers 519R and357F (5′-CTCCTACGGGAGGCAGCAG-3′) under the same re-action and cycling conditions as before with the exception ofthe substitution of one unit of Promega Taq polymerase and10X Promega PCR buffer B. PCR products were purified us-ing Genemate PCRpure Spin columns (ISC, Kaysville, UT)and sequenced with the Big Dye Terminator cycle sequenc-ing kit (Perkin-Elmer) according to the manufacturer’s proto-col. Sequence homology to known sequences was determinedby BLAST analysis (Altschul et al. 1990).

Statistical Methods. Differences between the microbiolog-ical results of two sampling media (i.e. core versus groundwater;dialysis chambers filled with water versus crushed basalt; coreversus crushed basalt-filled dialysis chambers) were tested withone-way analysis of variance (ANOVA) with significance de-termined at the p = 0.05 level. In a limited number of casesfor comparisons of the two types of dialysis chambers wherethe requirements of the test were met, a two-tailed paired t-testwas used to test for significant differences at the p = 0.05 level.Unless otherwise noted, values for all microbiological measure-ments are reported as means ± one standard deviation.

RESULTS

Quality of Core SamplesFor both boreholes, there was a one to two order of magnitude

reduction in concentrations of added tracers between the exte-rior and interior (portion analyzed) surfaces of the core (datanot shown). In a comparison of the microbiology of TAN-37cores with groundwater samples bailed from the borehole im-mediately following coring, there were distinctive bacterial taxarecovered only from the core samples and eukaryotic signaturefatty acids (18:2ω6; 18:3ω3) observed that were unique to thebailed groundwater samples (S. P. O’Connell, R. M. Lehmanand F. S. Colwell. Abstr. 98th Gen. Meet. Am. Soc. Microbiol.,abstr. N-18, 1998). Analysis of field-processed negative controlcores (previously combusted) and positive controls (spiked witha methanotrophic consortium) and the absence of organisms inthe disinfected drilling fluid indicated that core handling proce-dures did not contribute significantly to contamination. Very lownumbers of microorganisms and activities that were observed inTAN-33 cores and sharply contrasted with higher numbers inthe TAN-37 cores (Tables 2 and 3) testified to the effectivenessof the core handling procedures.

Effectiveness of Extracting Viable Cells from Crushed Basaltfor CLPP. The aqueous basalt extracts (set 1) from three dial-ysis chambers respired 55 ± 4 sole carbon sources of the 95substrates in the GN microplates, while the basalt particles re-maining after the extraction (set 2) respired 32 ± 7 sole carbonsources. Two of the extracted particulate basalt samples showedrespiration of a single additional substrate (methyl pyruvate)that was not seen with aqueous extracts, while the third sam-ple did not show respiration of any additional substrates. Theunextracted basalt particles (set 3) incubated in microwells con-taining substrates that were not respired by the communities inthe aqueous extracts (ca. 35 substrates) showed respiration ofthree, one and zero additional substrates for the three samples.Methyl pyruvate was again the additional respired substrate intwo of the three samples. No reduction of the tetrazolium salt wasobserved when sterile basalt particles with PBS were incubatedin the microwell containing methyl pyruvate. These results sug-gest that the method used for extraction of viable cells from thebasalt produces a representative sample of the attached com-munity, at least in terms of those organisms possessing mea-surable dehydrogenase activity in the Biolog GN microplateenvironment.

Comparison of Core and Groundwater Samples from WellTAN-33. Core samples collected from the less contaminatedwell, TAN-33, were nearly devoid of measurable cells or ac-tivities (Table 2). Total PLFA for the core samples was 1.4 ±1.4 µmol g−1 (n = 19), a value that was not significantly differentfrom zero. Dissimilatory iron-reducing bacteria were enrichedfrom about 25% of the cores, and phenol oxidizers were enu-merated in fifteen of the nineteen cores. Aerobic and anaerobicacetate mineralization in core samples were nearly undetectableafter three days of incubation (Table 2). After an extended incu-bation period of 14 days, 19.8 ± 21.5% and 6.7 ± 8.9% of theadded label were mineralized under aerobic and anaerobic con-ditions, respectively. The high standard deviations associatedwith these mean 14-day activity values indicated a strong vari-ability among samples that was reinforced by the relatively lowmedian values for acetate mineralization after a 14-day incuba-tion period (13.6% for aerobic and 1.7% for anaerobic). In fact,the means of all samples for 14-day aerobic and anaerobic ac-etate activities were not significantly different from zero (1-wayANOVA), and, in many cases, only one or two of the triplicatevials for a given sample showed a strong response. In compari-son, microbiological parameters for each of the TAN-33 ground-water samples was low, but usually measurable, partly due to en-hanced detection limits afforded by inoculation using undilutedgroundwater samples (Table 2). Community metabolic richness,a measure that is somewhat independent of sample mass, wassignificantly greater for groundwater than core samples (p <

0.05).Comparison of Basalt- and Water-Filled Dialysis Chambers

Incubated in well TAN-33. Considering the enumerations interms of ml of water and wet g of crushed basalt in the two dial-ysis chamber types, there was no significant difference between

176 R. M. LEHMAN ET AL.

Table 2Summary of well TAN-33 microbiology

Parametera Coreb Groundwater Dialysis—basalt Dialysis—water

Total cells (log) nd 4.97 (4.38)3/3

[4.84–5.07]

6.63 (6.25)7/7

[5.89–6.81]

6.44 (6.19)13/13

[6.11–6.80]

Aerobic heterotrophs (log) bd (<100)0/5

nd 5.63 (4.84)6/6

[5.57–5.69]

4.95 (4.56)6/6

[4.79–5.20]

Heterotrophic morphotypes bd (<1)0/5

nd 41 (9)6/6

[32–57]

41 (4)6/6

[38–48]

Phenol oxidizersc 114 (90)15/19

[<2–323]

0.1 (0.1)1/5

[<0.2–0.2]

14 (28)21/32

[<3.6–149.4]

23 (108)32/32

[0.36–617]

Methanotrophsc bd (<2)0/19

0.2 (0.1)4/5

[<0.2–0.4]

bd (<3.6)0/32

0.15 (0.24)11/32

[<0.36–0.92]

Propanotrophsc bd (<2)0/19

0.4 (0.4)4/5

[<0.2–0.8]

nd nd

Ammonium oxidizersc bd (<2)0/19

bd (<0.2)0/5

nd nd

H2-oxidizersc nd nd >1,00032/32

>10032/32

Iron-reducers 26%5/19

0%0/5

13%4/32

19%6/32

Metabolic richness 0.5 (1.0)6/19

[0–3]

24 (6.4)5/5

[17–33]

60 (8)32/32

[46–79]

80 (4)32/32

[73–88]

Aerobic mineralization 1.0% (2.6)17/19

[<0.1–11.1%]

nd 23.5% (1.8)32/32

[18.5–26.5]

nd

Anaerobic mineralization 0.1% (0.3)2/19

[<0.1–1.3%]

nd 34.2% (10.2)32/32

[18.2–74.5%]

nd

aEnumerations are reported per ml of groundwater or per wet g of rock. Iron-reducers are reported as the percentof samples which had positive enrichments (at least one of three triplicate enrichments). Metabolic richness is thenumber of sole carbon sources oxidized by the whole sample inoculated into Biolog GN microplates. Aerobic andanaerobic mineralization are reported in percent of added 14C-labeled acetate that was mineralized after 3 days.

bData are reported as follows: on the first line, the mean with one standard deviation in parentheses; on the secondline, the number of independent samples with a positive assay result over the the total number of independentsamples analyzed; on the third line, the range of measured values. Measurements below method detection limitswere assumed to be zero for the purpose of mean and standard deviation calculations. nd = no data. bd = belowdetect with method detection limit following in parenthesis.

cMost-Probable-Number (MPN) enumerations; for core samples, the MPN matrix contained six dilution levelswith five vials per level; for groundwater samples, the MPN matrix contained four dilution levels with three vialsper level; for dialysis chamber samples, the MPN matrix contained three dilution levels with three vials per level.

the two sample types in the number of total cells, heterotrophicmorphotypes, or phenol oxidizers (Table 2). In contrast, sig-nificantly more aerobic heterotrophs were recovered from thecrushed basalt compared to the water (p < 0.001, two-tailed,paired t-test) and significantly less metabolic richness was ob-

served in the basalt than in the water (p < 0.001, two-tailed,paired t-test). Substantial numbers of aerobic H2-utilizing bac-teria were present in both sample types; however, the dilutionswere not carried out far enough to determine if there was adifference between the two sample types. Principal components

SUBSURFACE MICROBIAL SAMPLING 177

Table 3Summary of well TAN-37 microbiology

Parametera Coreb Groundwater

Total cells (log) nd 5.35 (4.79)3/3

[5.23–5.49]

Aerobic heterotrophs (log) 6.83 (7.28)19/19

[3.56–7.92]

nd

Heterotrophic morphotypes 8.3 (2.2)4/4

[5–10]

nd

Phenol-oxidizersc 6223 (15,592)21/23

[<2–64,841]

15 (9)3/3

[5–24]

Methanotrophsc 22 (55)8/23

[<2–230]

19 (8)3/3

[9–24]

Propanotrophsc 958 (1976)18/23

[<2–7934]

1.1 (0.7)3/3

[0.36–1.5]

Ammonium-oxidizersc 1.2 (5.6)1/23

[<2–27]

8.6 (6.8)3/3

[1.5–15]

Iron-reducers 74%17/23

33%1/3

Metabolic richness 60 (19)23/23

[8–83]

82 (2)3/3

[81–84]

Aerobic mineralization 16.7 (4.0)23/23

[11.6–25.3]

nd

Anaerobic mineralization 29.2% (14.2)20/23

[<0.1–42.5%]

nd

PLFAd 1.6 (0.9)21/23

[<1–3]

0.25 (0.07)2/2

[0.2–0.3]

aEnumerations are reported per ml of groundwater or per wet g ofrock. Iron-reducers are reported as the percent of samples which hadpositive enrichments (at least one of triplicate enrichments). Metabolicrichness is the number of sole carbon sources oxidized by the wholesample inoculated into Biolog GN microplates. Mineralization is re-ported in percent of added 14C-labeled acetate that was mineralizedafter 3 days. PLFA is reported as total picomoles per g or ml.

bData are reported as follows: on the first line, the mean with onestandard deviation in parentheses; on the second line, the number ofindependent samples with a positive assay result over the the total num-ber of independent samples analyzed; on the third line, the range ofmeasured values. Measurements below method detection limits wereassumed to be zero for the purpose of mean and standard deviationcalculations. nd = no data.

cMost-Probable-Number (MPN) enumerations; for core samples,the MPN matrix contained six dilution levels with five vials per level;for groundwater samples, the MPN matrix contained four dilution lev-els with three vials per level.

d PLFA for groundwater from Kent Sorenson (unpublished data).

Figure 4. Principal components plot of the factor scores ofthe community carbon source utilization patterns from all of thedialysis chamber samples (n = 64 total: 32 basalt-filled cham-bers and 32 water-filled chambers). Factor 1, which accountsfor 39.6% of the variance in the data set (in parentheses), segre-gates basalt-filled dialysis chambers from their counterparts inthe water-filled chambers. Factor 2, which accounts for 8.9% ofthe total variance, indicates the variation within the two sampletypes, primarily the basalt-filled chambers.

analysis of the patterns of community sole carbon source utiliza-tion demonstrated a strong differences between the water-filledchambers versus those in the basalt-filled chambers (Figure 4).

Basalt- and water-filled chambers from the two depth inter-vals (103 and 113 m) were more intensively studied. FAMEprofiles were obtained on all morphologically distinct aero-bic, heterotrophic isolates (n = 76) recovered from these dial-ysis chambers. Considering the isolates that matched the MIDIdatabase, there appeared to be no segregation in identities be-tween the crushed basalt- and water-filled chambers. Gram-negative and gram-positive organisms were present in bothchamber types and common genera included Pseudomonas,Burkholderia, Brevundimonas, Acidovorax, Hydrogenophaga,Xanthobacter, Alcaligenes, Aurobacterium, Flavobacterium,Rhodococcus, Rhodobacter, Nocardia, Paenibacillus, and Mi-crococcus. Principal components analysis of FAME profiles wasused to compare all the isolates, including those that didn’t matchthe MIDI database, from the basalt- and water-filled chambersfrom the two depth intervals; again, no distinction between thetwo sample types was observed (Figure 5).

Similar banding patterns were observed in the DGGE anal-yses of basalt- and water-filled dialysis chambers from the 103and 113 m depths, with 9 to 13 bands observed in each sam-ple (Figure 6). Altogether, 21 unique bands that appeared inone or more of the samples were sequenced and BLAST anal-yses against the GenBank database indicated that the major-ity of the bands were most closely related to members

178 R. M. LEHMAN ET AL.

Figure 5. Principal components plot of the factor scores of thefatty acid profiles (mole percent) for isolates (n = 76) recoveredfrom basalt- and water-filled dialysis chambers incubated at the103 m and 113 m depths. The plot of Factor 1 (12.0%) and Factor2 (10.8%) did not distinguish isolates recovered from the basalt-(B) and water-filled (W) chambers.

of the Proteobacteria, Firmicutes, or Cytophaga-Flexibacter-Bacteroides groups. Examination of the presence or absence ofthese bands in each of the four samples shows that only one band(G) was unique to basalt samples and one band (U) was uniqueto water samples (Table 4).

Comparison of TAN-33 Core Samples with Crushed BasaltIncubated in Dialysis Chambers. The microbiology of theTAN-33 core samples was markedly different than that of thesame basalt, crushed and incubated in the open well in dialysischambers (Table 2). Far greater numbers and types of aerobicheterotrophs were recovered from the dialysis chambers, andcommunity metabolic richness was also significantly higher inthe dialysis chamber samples versus the core samples. Aerobicacetate mineralization for the crushed basalt from the cham-bers was relatively uniform throughout the array of 32 samples(23.5 ± 1.8%). In comparison, acetate mineralization observedin the core samples was minimal after the same 3-day incubationperiod, and only proceeded to measurable values in a few sam-ples after a 14-day incubation period. Interestingly, anaerobicmineralization (34.2 ± 10.2%) was higher than aerobic miner-alization in the dialysis chambers containing crushed basalt sam-ples, and this 3-day value was significantly greater (p < 0.001,1-way ANOVA) than even the corresponding 14-day value forcore samples (6.7 ± 8.9%). There were no significant differencesbetween the core and dialysis chambers in terms of numbers ofphenol oxidizers and methanotrophs. When the microbiologi-cal comparisons were limited to only depth-paired (collected <

0.3 m apart) core and basalt-filled dialysis chamber samples, thechamber samples were again observed to be markedly enrichedwith notably less data variation between replicate samples thanthe core samples (n = 8 paired observations, data not shown).

Figure 6. Comparative DGGE profile analysis of bacterial 16SrDNA fragments (nt 357-518 E. coli numbering) from dialy-sis chambers incubated in well TAN-33. Lanes: 1, basalt-filledchamber incubated at 103 m; 2, water filled chamber incubatedat 103 m; 3, basalt-filled chamber incubated at 113 m; 4, water-filled chamber incubated at 113 m. Letters indicate unique bandschosen for analysis.

Comparison of TAN-37 Core and Groundwater Samples.TAN-37 core had very high numbers of culturable aerobic het-erotrophs that even exceeded the number of total cells found inthe TAN-37 groundwater samples (Table 3). With two excep-tions, means of all microbial parameters were greater in the corethan in the groundwater, although the differences between meanvalues were statistically insignificant. The two exceptions werethat groundwater samples had significantly greater numbers ofammonium oxidizers and higher community metabolic richnessthan the core samples (p < 0.05). The cores had a mean PLFAvalue of 1.6 pmol g−1 that was significantly greater than zero(p < 0.05). The patterns of major classes of PLFA were verysimple and dominated by normal saturated PLFA with smallamounts of monounsaturated, terminally branched saturates, andpolyunsaturated PLFA (data not shown). The patterns were sim-ilar among the samples and further interpretation is constraineddue to the low biomass.

DISCUSSIONThe data on the authentic groundwater and core samples from

TAN-33 and TAN-37 indicate that (i) there was little detectablebiomass and activity in the fractured basalt and groundwatercollected from the mildly impacted (by the mixed waste plume)TAN-33; (ii) there was a general increase in biomass and activityin both core and groundwater samples from the more impacted

SUBSURFACE MICROBIAL SAMPLING 179

Table 4Presence of DGGE bands in 103 and 113 m dialysis cell samplesa

BANDb Basalt 103 m Water 103 m Basalt 113 m Water 113 m Sequence matchc

a X X X X alpha-Proteobacteria(Rhodopseudomonas palustris)

b X (−) (−) X Firmicutes

c X X X X Firmicutes; Actinobacteria(Arthrobacter chlophenolicus)

d X (−) X X Firmicutes; Actinobacteria(Agromyces cerinus)

e X (−) X X alpha-Proteobacteria

f (−) X (−) (−) Firmicutes; Bacillus/Clostridium(Clostridium thermopalmarium)

g X (−) X (−) beta-Proteobacteria

h X X X X Cytophaga-Flexibacter-Bacteroidesmany environmental clones

I X X X X gamma-Proteobacteria(Marinobacter articus)

j X (−) X X beta-Proteobacteria

k (−) X (−) (−) beta-Proteobacteria(Aquabacterium sp. Aqua3)

l X X X (−) No Match

m X (−) X X Cytophaga-Flexibacter-Bacteroides

n X (−) (−) X No Match

o X X X X Cytophaga-Flexibacter-Bacteroides

p X X X X No Match

q X X X X Cytophaga-Flexibacter-Bacteroides(uncultured Cytophagales clone MPD-4)

r X X X X No Match

s X X (−) X No Match

t (−) (−) X (−) gamma-Proteobacteria(Pseudomonas fluorescens)

u (−) X (−) X gamma-ProteobacteriaPseudomonas spp.

aPresence of band for that sample noted by an “X”; absence of band noted by “(−).”bBAND designation corresponds to alphabetical label applied to each band in Figure 6.cSequence identification was obtained by comparison with GenBank database using BLAST. Due to the short sequence amplified

from the bands, there were a number of close matches; therefore, group identifications are reported with an example of a high matchingsequence.

TAN-37 location, and numerous organisms were present thatmay engage in degradation of TCE under oxic and suboxic con-ditions; (iii) some evidence existed to support differences in themicrobiology of core and groundwater sample at both locations;and, (iv) there was high variation in the values for individual mi-crobiological properties measured on the TAN-37 core samples.

At the less-contaminated TAN-33 site, a greater number ofpositive responses were observed in assays performed on

groundwater samples compared to core samples, although theuse of undiluted groundwater for inoculum afforded lower assaydetection limits. All of the TAN-33 groundwater samples werepositive for 2 or more assays, while only 5 of the 19 core sam-ples had positive values for 2 more assays. Several core samplesdid not exhibit positive responses to any of the assays. A simi-lar weak microbiological response was observed in core samplescollected from another fractured rock aquifer in comparison with

180 R. M. LEHMAN ET AL.

adjacent groundwater samples (Lehman et al. 2001). Increasedorganic contamination at the TAN-37 site probably enhancedbiomass, particularly that associated with the core samples. AllTAN-37 groundwater samples were positive for all parameters,but the highest enumeration recorded for a single physiologicalgroup was 24 cells ml−1.

Conversely, core samples were quite variable with valuesranging from 0 cells g−1 up to 105 cells g−1 phenol oxidizers,104 cells g−1 propanotrophs, and 102 cells g−1 methanotrophs.Only 8 of 23 cores were positive for methanotrophs and just 1of 23 cores was positive for ammonium oxidizers. While meanparameter values were often not statistically different betweencore and groundwater samples, core samples exhibited decid-edly higher maximal values and greater variability in responsescompared to groundwater samples. There are some reports onscales of variability in the microbiology of porous media sam-ples (Brockman and Murray 1997) and groundwater samples(Franklin et al. 1999), but a noticeable lack of data from aquiferssimilar to fractured basalt or comparisons of both geologic mediaand groundwater samples. Despite the relatively high number ofcores studied at site TAN-33 (19) and TAN-37 (23), it will requirean even higher sample number to capture the true extent of mi-crobiological variability in fractured rock aquifers and to makeeffective statistical comparisons. It should be noted that surfacesfrom highly conductive macroscopic fractures and rubble zonesin the aquifer were not sampled. These surfaces are certain to becolonized by bacteria, perhaps phylogenetically distinct or pos-sessing different metabolic activities than bacteria in the basaltmatrix, but no method was devised to aseptically-sample thesefracture surfaces.

The evidence for asymmetric distribution of microorganismsor their activities between attached and free-living states pre-sented in this study reinforces similar findings from controlledcomparisons conducted in unconsolidated sedimentary aquifers(Kolbel-Boelke et al. 1988; Godsy et al. 1992; Bekins et al.1999) and a fractured rock aquifer (Lehman et al. 2001). In-creased organic contamination at the TAN-37 site seems tohave favored increased biomass associated with core samples,and perhaps the culturability of microorganisms suspended inthe groundwater. This observation contrasts with previous re-ports suggesting that elevated nutrient and carbon concentrationsincrease partitioning to the aqueous phase (Harvey et al. 1984;Hazen et al. 1991). The differences between attached and free-living aquifer bacterial communities observed in the current andprevious studies may be attributed to either differences in com-munity composition or functional expression (or both). In all ofthese reports, the comparison of core and groundwater samplesis largely based on growth dependent assays and therefore rep-resent a comparison of community functional potential and, toa lesser extent, community composition. In the current study,growth independent assays that were intended to describe thecommunity composition of the core and groundwater samplesgenerated limited results. Total PLFA for most samples was ator just above the method detection limit (ca. 1.0 ρmol g−1).

The patterns of major classes of PLFA were all very simple andsimilar between samples and therefore prevented further inter-pretation. Extraction of DNA was attempted from TAN-33 andTAN-37 cores and the quantity of the DNA and/or its suitabilityfor PCR-amplification were poor and very minimal results wereobtained. The limitation of culture-independent methods (e.g.,DNA, PLFA) applied to low biomass, fractured rock core hasbeen previously encountered (Lehman et al. 2001).

In TAN-33, the basalt- and water-filled dialysis chamber sam-ples were greatly enriched compared to the authentic core andgroundwater samples. The surface area available for coloniza-tion cannot account for the higher biomass in the basalt-filledchambers compared to the cores because the total surface area(as measured by N2 (g) adsorption) for crushed basalt was onlymodestly (ca. 2×) greater than that of the intact cores (T.L.McLing, personal communication). This modest increase in to-tal surface area of the basalt upon crushing reflects a relativelysmall increase in external surface area compared to the largeamount of internal surface area present in the basalt. The moredetailed data on the basalt- and water-filled chambers from the103- and 113-m depths indicates that the recovered communi-ties were compositionally similar and that the composition ofthe sampled community appeared independent of the inclusionof an attachment substratum (i.e., basalt). There was no differ-ence in the identities of the aerobic chemoheterotrophic isolatescultured from these two types of chambers, there was high sim-ilarity in their 16S rDNA sequence profiles, and only two 16SrDNA sequences were unique to either the basalt- or water-filledchambers. However, the CLPP results support clear differencesin community functional potential. It would be revealing to learnif this same trend was true for the core and groundwater samples;however, the numerous negative responses and limitations ofthe culture-independent methods preclude conclusions regard-ing the community composition of the authentic aquifer samples.

A number of researchers have used artificial substrata in-cubated in wells to sample or make measurements upon theattached subsurface communities (Hirsch and Rades-Rohkohl1990; Holm et al. 1992; Pedersen and Ekendahl 1992; Pedersenet al. 1996; Alfreider et al. 1997). Disturbance of the local aquiferduring drilling (or mining) and the subsequent artificial environ-ment of the well (or mine) represent obvious changes in condi-tions that may influence colonization of substrata incubated ina well (Thomas et al. 1987; Hirsch and Rades-Rohkohl 1988;Alfreider et al. 1997). Whether these artificial substrata obtain arepresentative sample of communities attached to the in situ ge-ologic substrata has not been demonstrated, although for somecomparative studies that issue may not be critical. The resultsof our study indicate that this in situ sampling device (dialysischambers) recovered more biomass and a much higher diversityof aerobic heterotrophs than detected in the authentic aquifersamples. The ability of artificial substrata to recover a greaterrange of the aquifer microbial diversity than actual samples maybe attractive for some studies. The influence of the composi-tion of the attachment substratum on recovered communities

SUBSURFACE MICROBIAL SAMPLING 181

and their relationship to that of authentic samples bears furtherinvestigation.

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