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was represented by terrestrial humic uorescence signatures, which are typically observed in surface

) has borld ben varionsabauettings

There are notable differences in DOM composition between sur-face water and groundwater. The dominant components of DOM inmost surface water environments are dissolved humic substances,primarily fulvic acids (Thurman, 1985). Relative to other types ofDOM, humic substances accumulate in surface water environ-ments because of their refractory nature (Frimmel, 1998). In con-trast, ca. 30% of the DOM in uncontaminated subsurface watersconsists of hydrophilic or neutral material not considered to be

carbon sources (e.g. bicarbonate) by gaining cellular energy fromoxidation/reduction reactions, such as oxidation of reduced sulfurcompounds (i.e. sulfur oxidation) or methanogenesis. Most terres-trial, chemolithoautotrophically-based ecosystems are associatedwith cave and karst terrains, whereby autochthonous OM is suf-cient to support entire ecosystems. Some notable systems includethe Movile Cave, Romania (Sarbu et al., 1996), the Frasassi Cavesystem in Central Italy (Vlasceanu et al., 2000) and Lower KaneCave in Wyoming, USA (Engel et al., 2004).

The CDOM properties of these cave waters are expected to bedifferent from those of oceans, estuaries and freshwater near the

* Corresponding author. Tel.: +1 225 578 1426; fax: +1 225 578 1476.

Organic Geochemistry 41 (2010) 270280

Contents lists availab

o

evE-mail addresses: jbirdw1@gmail.com, jbirdwell@usgs.gov (J.E. Birdwell).is primarily derived from terrigenous and aquatic macro (e.g.plants, animals) and microorganisms (e.g. algae, phytoplanktonand bacteria). Chromophoric dissolved organic matter (CDOM),the fraction that absorbs ultraviolet (UV) and visible light, is thecontrolling factor for the optical properties of surface waters(Green and Blough, 1994). Spectroscopic techniques can provideinformation about the source and composition of the DOM presentin a system at natural abundance concentration, thereby eliminat-ing the need for isolating or concentrating it prior to analysis (Co-ble, 1996; Hudson et al., 2007).

(terrigenous) humic substances in subsurface waters, includingretention of terrestrial DOM by the soil column as water percolatesinto the subsurface, biotic molecular transformation of terrigenousDOM input (Einsiedl et al., 2007), or in situ DOM production by mi-crobes indigenous to subsurface environments.

Reactive mineral surfaces and energetically rich waters supportan array of chemolithoautotrophic microorganisms in subsurfacehabitats (e.g. Stevens, 1997; Kinkle and Kane, 2000). When theallochthonous carbon input is limited or lacking altogether, thesemicrobes can provide a source of organic carbon from inorganic1. Introduction

Dissolved organic matter (DOMgated in water systems around the wroles this ubiquitous material plays iecological processes (Findlay and Si2004; Judd et al., 2006). In natural s0146-6380/$ - see front matter Published by Elsevierdoi:10.1016/j.orggeochem.2009.11.002waters, as well as soil and sediment porewaters. Comparison of the cave and spring waters with a widearray of reference humic substances and OM from other environments showed a continuum of spectralproperties constrained by origin and degree of humication.

Published by Elsevier Ltd.

een intensely investi-cause of the signicantus biogeochemical andgh, 2003; Anesio et al.,, DOM parent material

recalcitrant, including polysaccharides, alkyl alcohols, aldehydes,ketones and amides (Leenheer, 1981). Based on research fromgroundwater (Leenheer and Noyes, 1984; Baker and Lamont-Black,2001) and cave drip waters (e.g. Baker and Genty, 1999), the uo-rescent amino acids generally attributed to microbial activity(tryptophan and tyrosine) account for most of the non-humicCDOM. Several processes may control the lack of surface-derivedfrom microbially-derived material, and the degree of OM humication was low. Little of the CDOM poolCharacterization of dissolved organic matabsorbance and uorescence spectroscop

Justin E. Birdwell a,*, Annette Summers Engel b

aCain Department of Chemical Engineering, Louisiana State University, Baton Rouge, LAbDepartment of Geology and Geophysics, Louisiana State University, Baton Rouge, LA 70

a r t i c l e i n f o

Article history:Received 1 June 2009Received in revised form 19 September2009Accepted 3 November 2009Available online 6 November 2009

a b s t r a c t

Chromophoric dissolved ortra from suldic cave and thave a limited allochthonoon chemolithoautotrophichad uorescence signatureindices and absorbance sp

Organic Ge

journal homepage: www.elsLtd.r in cave and spring waters using UVVis

03, USA, USA

ic matter (CDOM) was examined using uorescence and absorbance spec-mal and non-thermal surface-discharging spring waters. Many of the sitesupply of organic matter (OM) and contain ecosystems that are dependentrobial communities. Water-extracted OM from microbial mats at the sitesnsistent with the uorescent amino acids. Based on uorescence-derivedal characteristics, the origin of the cave and spring CDOM appeared to be

le at ScienceDirect

chemistry

ier .com/locate /orggeochem

OM and humic substances. Our assessment of these waters pro-vides a distinctive and novel array of CDOM signatures con-

Geostrained by microbial inuences that can serve as a comparisonfor future water resource investigations and carbon budgetstudies. From a management and conservation perspective,knowledge of CDOM characteristics and sources is critical forpreserving groundwater quality and ecosystem integrity, espe-cially for systems that may be highly susceptible to contamina-tion, like karst aquifers that supply a signicant portion of theworlds drinking water (Spizzico et al., 2005; Green et al., 2006;Birdwell and Engel, 2009).

2. Background

2.1. Fluorescence spectroscopy

Fluorescence spectroscopy is a highly selective technique foranalyzing organic substances because only those compounds con-taining moieties with conjugated bonds are observed (Lakowicz,1999). The method is also highly sensitive at natural abundancelevels and analyte concentration through isolation is generallynot needed (Coble, 1996). There are two major categories of uoro-phores in uncontaminated natural waters: humic-like and protein-like (Baker and Lamont-Black, 2001; Chen et al., 2003). Humic-likeuorescence is a term used to describe spectral features thatresemble those of isolated humic and fulvic acids (Alberts andTakcs, 2004), while the term protein-like uorescence describespeaks that are attributed to the uorescent amino acids tryptophanand tyrosine (Yamashita and Tanoue, 2004). Both humic-like andprotein-like uorophores absorb, and are excited by, UV light(240280 nm). Humic-like uorophores emit primarily in the vio-let to near-green (400500 nm), which results from the presence ofquinone-like structures sourced from the degradation of terrestrialbiomaterial such as lignin (Ariese et al., 2004; Cory and McKnight,2005). In contrast, protein-like uorophores emit in the UV tonear-violet (300380 nm). These starkly different humic-like andprotein-like uorescence characteristics allow the selective andsurface, or soil and sediment porewaters, because of their uniquebiogeochemical conditions and lack of exposure to sunlight. How-ever, our current understanding of microbially-derived DOM insubsurface ecosystems, which could account for a signicant frac-tion of the DOM in the subsurface (e.g. Whitman et al., 1998), isincomplete because few studies, apart from those of material inthe open ocean (e.g. Yamashita and Tanoue, 2003), have been con-ducted on the CDOM from systems that are not signicantly inu-enced by terrigenous DOM sources or photosynthetically-drivenmicrobial activity. Photosynthetically-driven microbial activityhas been shown to produce humic-like substances in the absenceof terrestrial, plant-based material like lignin (Moran and Hodson,1990; Claus et al., 1999; Ogawa et al., 2001; Hertkorn et al., 2002)and is also the primary source of DOM in Pony Lake and Lake Fryx-ell in Antarctica (e.g. Aiken et al., 1996; McKnight et al., 2001; Ful-ton et al., 2004).

In this study, the UVVis spectroscopic properties of CDOMfrom a variety of cave and spring waters were investigated,including suldic karst springs and several geothermal, non-karstsprings. The sites are important microbial habitats (e.g. Sarbuet al., 1996; Vlasceanu et al., 2000; Engel et al., 2004, 2008; Porterand Engel, 2008). Our goal was to determine how the uores-cence and absorbance characteristics of water and microbiologicalmaterial from these systems compare with each other and withCDOM collected from surface environments, including isolated

J.E. Birdwell, A.S. Engel / Organicsensitive distinction of the relative contributions of natural mate-rials in DOM (e.g. Coble, 1996; Chen et al., 2003; Hudson et al.,2007) that possibly reect different origins (e.g. McKnight et al.,2001; Huguet et al., 2009) and various stages of humication ordiagenesis (Zsolnay et al., 1999).

There are a number of different methods for interpreting uo-rescence data, from peak picking to complex numerical modelingschemes like Parallel Factor Analysis (e.g. Coble, 1996, 2007; Sted-mon et al., 2003; Stedmon and Markager, 2005; Cory andMcKnight, 2005; Hunt and Ohno, 2007; Murphy et al., 2008; Cooket al., 2009). Excitationemission matrix (EEM) uorescence spec-troscopy uses an assembly of uorescence emission spectra col-lected over a range of excitation wavelength to summarize theentire steady-state UV and visible uorescence behavior of a par-ticular sample (Fig. 1). EEM spectra provide information on the rel-ative intensity of uorescence at different excitationemissionwavelength pairs (or regions) and the Stokes shifts of uorophoresin a way that is fast and convenient, particularly for complex mix-tures of uorescent components (Coble, 1996). The typical regionof the UV and visible uorescence spectrum obtained on DOMsamples is from the mid-UV (or UVC, >200 nm) to the violet andnear-blue (ca. 450 nm) in the excitation wavelength (kEx), andemission wavelengths (kEm) from mid-UV to the yellow or near-or-ange (ca. 600 nm). In general, little CDOM uorescence emission isobserved past kEm ca. 550 nm (Smith and Kramer, 1999). Coble(1996, 2007) identied a set of characteristic CDOM descriptivepeaks in EEM uorescence spectra. These peaks have been ob-served in spectra collected from ltered surface waters, as wellas isolated humics and OM samples. There are three commonly ob-served humic-like peaks (Fig. 1): UVC-excited (designated A; kEx240260 nm, kEm 400460 nm), UVA-excited (designated C; kEx320360 nm, kEm 420460 nm) and marine humics (designatedM, kEx 290310 nm, kEm 370410 nm), though peak M has alsobeen attributed to coastal and marine biological activity (Parlantiet al., 2000), anthropogenic input to natural waters (Stedmonand Markager, 2005) and has been proposed as a precursor forpeak C uorophores (Burdige et al., 2004). Peaks indicative of bio-logical activity or protein-like material include tyrosine-like peaks(B, kEx 270280 nm, kEm 300315 nm) and tryptophan-like peaks(T, kEx 270280 nm, kEm 345360 nm).

2.2. Fluorescence-derived indices

Fluorescence intensity ratios can be used to infer the relativecontributions from autochthonous and allochthonous OM in natu-ral waters. McKnight et al. (2001) found that the ratio of the emis-sion intensity at kEm 450 nm to that at kEm 500 nm, followingexcitation at kEx 370 nm, provides a metric for distinguishingCDOM derived from terrestrial and microbial sources. This ratiois referred to as the uorescence index (FI; Fig. 1). FI has been cal-ibrated using data collected on fulvic acids and whole water sam-ples obtained from environments known to contain CDOM derivedfrom either allochthonous or autochthonous sources (McKnightet al., 2001). FI values can be inuenced primarily by the intensitiesof the UVA-excited humic peak (C), peak M, and possibly other, asyet unidentied, uorophores with large (>80 nm) Stokes shiftsthat emit in the bluegreen region of the visible spectrum (Burdigeet al., 2004). FI values of 1.4 or less indicate DOM of terrestrial ori-gin and values of 1.9 or higher correspond to microbially-derivedmaterial. An inverse relationship exists between FI and DOM aro-maticity, as determined from CPMAS 13C NMR (McKnight et al.,2001) and FI increases as the C/N ratio decreases (Wolfe et al.,2002). Terrestrial organic compounds, particularly lignin, are ex-pected to contain more conjugated aromatic structures thanmicrobially-derived substances and terrestrially-derived humicsubstances should have greater emission intensity at longer

chemistry 41 (2010) 270280 271wavelength as a result of the increase in the C/H ratio during dia-genesis (Stevenson, 1982). Consequently, the ratio of short (kEm450 nm) to long wavelength (kEm 500 nm) emitting uorophores

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Fig. 1. Summary of CDOM characteristics of natural water samples. EEMs for threelines represent tyrosine, solid lines tryptophan, and dashed lines Suwannee River FA, arepresent the Coble (1996, 2007) peak designations A, C, M, T and B (see Section 2 folines, kEx 254 nm), BIX (dark gray points, kEx 310 nm) and FI (light gray points, kEx 3scattering bands (solid) and position where ground level solar radiation intensity be

272 J.E. Birdwell, A.S. Engel / Organicfrom terrestrial sources.Another index was proposed recently to assess the relative con-

tribution of autochthonous DOM in water samples, called the bio-logical/autochthonous index or BIX (Huguet et al., 2009). Like FI,BIX is calculated from the ratio of emission intensities at a shorter(kEm 380 nm) and longer (kEm 430 nm) wavelength using a xedexcitation (kEx 310 nm; Fig. 1). Formulation of BIX was inuencedby the presence of two common peaks in uorescence spectra col-lected on surface waters, attributed to terrestrial and microbialcomponents. Peak M is at the shorter emission wavelength (Coble,1996) (Fig. 1) and is attributed to autochthonous DOM production(Parlanti et al., 2000; Burdige et al., 2004; Huguet et al., 2009). Thelonger emission wavelength peak is greatly affected by the nearbyUVA-excited terrestrial humic peak C, which is considered an indi-cator of allochthonous carbon in aquatic systems (Coble, 1996,2007) or a more diagenetically altered form of peak M uorophores(Burdige et al., 2004). Values of BIX between 0.8 and 1.0 corre-spond to freshly produced DOM of biological or microbial origin,whereas values below ca. 0.6 are considered to contain littleautochthonous OM.

The degree of DOM humication is an indicator of a materialsage and recalcitrance within a natural system (Zsolnay et al.,1999; Ohno, 2002). Highly humied organic substances are gener-ally resistant to degradation and are expected to persist in theenvironment longer than substances with a low degree of humi-cation. Emission uorescence has been used to estimate the degreeof humication of DOM extracted from soils and other OM sourcesusing various spectral analyses. Zsolnay et al. (1999) proposed ahumication index (HIX) determined from the ratio of two inte-grated regions of an emission scan (sum from kEm 435480 nm di-vided by the sum from kEm 300345 nm) collected with excitationat 254 nm as a method for comparing the relative humication ofDOM samples (Fig. 1). HIX is low ( ca. 300 nm).

chemistry 41 (2010) 270280of decomposition (Hunt and Ohno, 2007; Wickland et al., 2007) orfractionation of DOM by sorption onto mineral surfaces (Ohnoet al., 2007). Water extractable DOM from soil and soil porewaterhas HIX values between 10 and 30 (Kalbitz et al., 2003; Wicklandet al., 2007). Huguet et al. (2009) applied the HIX to estuarine sam-ples across a salinity gradient and found that it generally decreasedwith increasing salinity (values ranged from 2 to 17). HIX isstrongly correlated with DOM aromaticity and inversely correlatedwith carbohydrate content (Kalbitz et al., 2003).

2.3. Absorbance spectroscopy

Absorption of UV and visible light by CDOM generally decreasesexponentially with increasing wavelength. This has been modeledin a number of studies (Stedmon et al., 2000; Kowalczuk et al.,2005; Murphy et al., 2008), using the following equation

ak akref eSekkref K 1where a(k) is the absorption coefcient at wavelength k (m1), krefthe reference wavelength (nm), Se the spectral slope parameter(nm1) and K, a background correction parameter to account forbaseline shift due to scattering (m1) (Stedmon et al., 2000; Kow-alczuk et al., 2005); a(kref) is proportional to dissolved carbon con-centration in waters containing similar types of chromophores(Stedmon et al., 2000). Se has been related to the ratio of fulvic tohumic acids and average molecular weight for oceanic CDOM(Twardowski et al., 2004).

3. Materials and methods

3.1. Sample acquisition and geochemical characterization

Water samples were collected from cave (aphotic) and spring(photic) environments, including suldic, non-suldic (freshwater)

imum

)

GeoTable 1Description of water and microbial mat samples and summary of uorescence maxindices.

Sampling site (Fig. 2 panel) Temp(C)

pH TDS(mg l1)

IMax(RU

Baker Hot Springs, WA, USAa (a) 36.2 8.2 548.6 1.49Big Sulphur Cave, KY, USAa,b,c (b) 15 7.2 NM 4.76

Frasassi Caves, ItalyResurgence Springa,c 13.8 7.4 1408 3.79Ramo Sulfureoa,b,c 13.6 7.1 1350 2.71Sulde surface wella,b,c (g) 13.7 7.4 1396 2.32Grotta Bellaa,b,c (h) 13.8 7.3 1402 2.16

Mat watersPozzo di Cristalia,b,c (m) 13.4 7.2 1903 3.08Sulde surface wella,b,c (n) 13.7 7.4 1396 3.15Grotta Bellaa,b,c (o) 13.8 7.3 1402 2.72

Glenwood Hot Spring, CO, USAa,b,c (c) 48 6.4 18,744 1.13Jemez Spring, NM, USAa 46.6 5.9 5264 2.04

Lower Kane Cave, WY, USAMat water (200 m)a,b,c (l) 22.5 7.5 393 4.48Water (63 m)a,b,c (e) 22.5 7.4 388 2.52Water (200 m)a,b,c 22.5 7.5 393 2.00

Pah Tempe Hot Springs, UT, USAMat watera (k) 445 6.9 NMd 8.08Alcove watera 445 6.9 NM 1.47

Sharon Springs, NY, USAa (d) 10 67 NM 7.16Sulfur Springs, IN, USAa,c 13.7 7.3 3876 1.85

J.E. Birdwell, A.S. Engel / Organicand geothermal systems (Table 1). Some of the spring waters thatwere exposed to sunlight upon reaching the surface were collectednear the source to limit the effect of solar irradiation. Each watersample was passed through a 0.45 lm Whatman glass ber lter(GF/F, autoclaved and precombusted at 500 C) and a 0.2 lm PTFE(Millipore, Bedford, MA) lter in tandem before storage in HDPEbottles. For some sites, raw lamentous microbial mats were col-lected with site water. Following centrifugation and ltration, themat waters (herein referred to as mat extracts) were diluted by1001000-fold to obtain solutions with UV absorbance (240 nm)of ca. 0.1 and to be consistent with the water samples. Filteredwater and mat extracts were stored on ice for transport and main-tained at 4 C until analysis. Depending on the circumstances ofcollection for each water sample, storage times varied between

(m). Eq. (1) was tted to absorbance data between 300 and

Geo600 nm, and 375 nm was the reference wavelength. The absorbancedata were tted using a non-linear regression technique, ratherthan the linearized form of Eq. (1), because the non-linear approachhas been found to yield smaller residuals (Kowalczuk et al., 2005).

Spectral corrections for primary and secondary inner lter ef-fects in the uorescence spectra were made using absorbance data(Lakowicz, 1999). Raman scattering was mitigated by subtracting ablank EEM spectrum collected on pyrogen-free deionized(>18.1 MX) water from each corrected EEM. Rayleigh scattering ef-fects were edited from each spectrum following correction andblank subtraction. FI, BIX, and HIX values were determined usingdata from the corrected EEM spectra.

A set of reference uorescence spectra was obtained to repre-sent CDOM from different surface environments, and compoundswhose uorescence characteristics are similar to CDOM spectralfeatures observed in other studies. Samples from the InternationalHumic Substances Society (IHSS) served as proxies for dissolvedhumic substances (humic and fulvic acids, HAs and FAs, respec-tively) derived from soil (Elliot HA, Pahokee HA, Wakish HA andFA), surface waters (Nordic HA, FA and NOM; Suwannee RiverHA, FA and NOM; Pony Lake FA) and coal (Leonardite HA). Addi-tional humic isolates included Amherst HA (soil), Laurentian HAand FA (soil), Aldrich HA (coal) and a series of sediment humic frac-tions extracted using 0.1 N NaOH from six sites in Louisiana, NewYork and Maryland. Porewater was extracted from the sedimentsamples by centrifugation and analyzed following ltration(0.45 lm GF/F, 0.2 lm PTFE). The reference humics and sedimentderived samples were used in a recent study of DOM isolated fromthe Atchafalaya Basin (Cook et al., 2009). Other standards includedL() and D(+) tryptophan (Acros Organics, Thermo Fisher Scientic,Inc., NJ, USA), L() and D() tyrosine (Acros Organics) and tryptone(Fisher Bioreagents, Fisher Scientic, Inc., NJ, USA) to obtain a pro-tein-like signature. All comparison samples were made or dilutedusing deionized water and prepared such that the concentrationswere ca. 10 mg organic carbon l1. The nal pH of the referencematerials was adjusted to between 6.5 and 7.0.

4. Results and discussion

4.1. General spectral features

Geochemical data for each of the samples are listed in Table 1.The EEM spectra of the cave and spring waters (Fig. 2, panels athrough i) contained many of the characteristic peaks observedin other studies of marine and terrestrial CDOM (e.g. Fig. 1). Thepeaks attributed to uorescent amino acids were most evident inthe spectra collected from the mat extracts (Fig. 2, panels jo).The spectra for the IHSS reference collection and other terrestrialand aquatic samples (data not shown) were composed primarilyintensities are normalized to the emission intensity of the deion-ized water Raman signal at kEx 348 nm, kEm 395 nm. UVVis absor-bance spectra were collected using a double beam UV-3101PCspectrophotometer (Shimadzu Corporation, Kyoto, Japan) and a1 cm quartz cuvette over the range 200600 nm with deionizedwater as the reference. To t the data to Eq. (1), optical density(absorbance value) was converted to absorption coefcient:

ak 2:303Akl

2

where A(k) is the optical density at wavelength k (dimensionless)and l the length of the cell used in the absorbance measurement

274 J.E. Birdwell, A.S. Engel / Organicof the UVC and UVA-excited humic peaks (A and C), as describedby Coble (1996). The maximum emission intensities observed forSuwannee River and Pony Lake FAs (SRFA and PLFA, respectively),considered to be representative end members for terrestrial (SRFA)and microbially-derived (PLFA) humic substances in surface waters(McKnight et al., 2001), were represented by peak A (for the rangeof wavelengths investigated) and their position differed by ca.15 nm (437.5 and 422.5 nm, respectively). The typical emissionmaxima for the cave and spring water samples were at the shortend of the wavelength range for peak A uorophores (kEm ca.400420 nm). The dominant protein-like peaks from mat extractswere attributed to tyrosine (kEm ca. 300 nm) and tryptophan (kEmca. 350 nm), corresponding to the peak B and T regions, respec-tively. The tryptone spectrum was dominated by tryptophan uo-rescence (peak T) and also contained a less intense tyrosine-likepeak (B). Corrected maximum uorescence emission intensity(IMax) for all waters from the caves and springs sampled was be-tween 1 and 10 RU.

The absorbance spectra for the cave and spring waters hadsteep drops in optical density between 200 and 250 nm, followedby an exponential decrease with increasing wavelength beyond280 nm. Many samples had a peak or shoulder in the ca. 260270 nm range, consistent with strong absorbance by uorescentamino acids, but could also be due to the presence of a wide rangeof other specic compounds that absorb in this region of the UVspectrum. Absorbance coefcients determined at 375 nm rangedfrom 1.50 (Glenwood Hot Springs, CO) to 8.50 m1 (El Tatio GeyserPool, Chile), with an average of 2.52 m1 (standard deviation1.51 m1). Spectral slopes determined using Eq. (1) for the watersvaried from 4.20 104 (Glenwood Hot Springs, CO, USA) to4.56 103 nm1 (El Tatio Geyser Pool), with an average slopefor all samples of 1.48 103 nm1 (std. dev. 8.95 104) andcoefcient of determination (R2) of P0.90. The humic and CDOMreference materials had a(375) values between 0.71 (NY harborsediment porewater) and 12.27 m1 (Elliot soil HA, IHSS), andspectral slopes between 7.30 103 (Pahokee Peat HA, IHSS) and1.68 102 nm1 (LA salt marsh sediment porewater), withR2P 0.98. Fig. 3 contains a set of reference UVVis absorbancespectra for various humic standards, as well as the spectrum col-lected for a sample from the Frasassi Cave system in Italy (RamoSurfureo).

4.2. Fluorescence indices

None of the water or mat extracts had FI values 1.9 for half of the samples, with three samples havingvalues > 2.2. These higher values are similar to those of lteredwhole water samples collected from a perennially ice-covered Ant-arctic lake (McKnight et al., 2001 and references therein) and thesamples with FI values between 1.6 and 1.9 are consistent with sig-nicant quantities of microbially-derived CDOM (e.g. McKnightet al., 2001), but the relative contribution of autochthonous andallochthonous material cannot be discerned from the FI values.All the coal, soil-derived and aquatic reference humic substanceshad FI values between ca. 1 and 1.3, with the exception of PLFA(1.51). The lowest value was for the Amherst soil HA (0.82). DOMextracted from sediment particles or present in sediment porewa-ters had values of ca. 1.75, consistent with material representing amixture of allochthonous and autochthonous sources. Tryptonehad a value of 2.45, higher than all but one mat extract (El TatioMain Geyser; FI 2.63).

BIX values for the cave and spring water samples were generally>0.7, the exceptions being all the hot (>30 C) springs and Big Sul-fur Cave (KY, USA), which had values between ca. 0.5 and 0.7. Themat samples had values of ca. 1 or higher. These high values for thecave and spring water samples are consistent with those observed

chemistry 41 (2010) 270280by Huguet et al. (2009) for autochthonous DOM derived frommicrobially-dominated samples. The reference humic substancesfrom coal, soil and surface waters demonstrated a wider range of

d S

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J.E. Birdwell, A.S. Engel / OrganicBIX than FI values (0.310.78), though >80% had BIX values 0.7. BIX for tryptone was higher (5.16) than all of the water sam-

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chemistry 41 (2010) 270280 275ples, although the highest value among the mat extracts was sim-ilar (El Tatio Main Geyser; BIX 4.92).

All the cave and spring waters had HIX values 2 (Baker Hot Springs, WA; Sulde SurfaceWell, Frasassi Caves, Italy). Such low values are consistent withthe majority of the CDOM being principally composed of fresh

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276 J.E. Birdwell, A.S. Engel / Organic Geochemistry 41 (2010) 270280biological detritus, similar to material derived from lysed cells ob-tained by fumigating soil samples with CHCl3 (Zsolnay et al., 1999)and fresh organic substances extracted from plant biomass andanimal manure (Ohno and Bro, 2006; Hunt and Ohno, 2007; Ohnoet al., 2007). The reference humics had much higher HIX valuesthan the cave and spring samples (HIX ca. 1060), consistent withthe materials representing isolated, recalcitrant fractions of DOM

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Fig. 3. UVVis absorbance spectra comparing terrestrial (Suwannee River FA and Lesample from the Frasassi Cave system in Italy (Ramo Sulfureo).from coal, soil and surface waters. The HIX values of the coal de-rived humics were the highest (>50). Soil and aquatic humicshad a wide range of values (1030 and 2050, respectively) andthe sediment-derived and porewater DOM samples were some-what less humied (HIX ca. 620), with the exception of one sam-ple (LA salt marsh porewater; HIX 28.37). Tryptone had a low value(0.05), which may or may not be meaningful, although it appears torepresent a wholly unhumied biological signature.

Table 2 provides a summary of the range of HIX, FI and BIX val-ues for particular types of CDOM examined in this study. For thewater samples, the low HIX and high BIX and FI values may repre-sent uorescent, water soluble, extracellular substances excretedby microorganisms, detritus resulting from cell death, and aquatichumic substances less humied than surface water humics, possi-bly due to a lack of exposure to solar radiation. Specically, pro-

Table 2CDOM categories determined from the literature and calculated from data provided.

Category (Ref.) FI range HIX range BIX range

Coal derived humics 1.01.2 >50 0.35Soil derived humics 0.81.3 1030 0.30.78Sediment OM 1.21.6 520 0.581.22Aquatic humics 1.01.3a 2050 0.300.40Soil porewater (Wickland et al., 2007) 1.21.5 525 NDb

Sediment porewater 1.51.8 530 0.571.10Soil amendments (Hunt and Ohno, 2007) 1.21.4 1.6 2 2.0Protein (tryptone) (this study) >2 (2.45)

Fluorescence Index1.0 1.5 2.0 2.5

Hum

ifica

tion

Inde

x

0

10

20

30

40

50

60 Coal derived humicsSoil derived humicsAquatic humicsTryptoneSediment OMSediment PorewaterSoil Porewater(Wickland et al., 2007)Soil Amendments(Hunt and Ohno, 2007)Cave and Spring watersMat ExtractsAquifer well and spring waters(Birdwell and Engel, 2009)

Pony Lakefulvic acid

Suwannee Riverfulvic acid

Fig. 4. Comparison plot of FI and HIX values for different CDOM types. Includes cave and spring waters and mat extracts, reference CDOM included coal, soil and surface water(aquatic) derived humics, sediment organic matter, sediment porewaters and a protein signature from tryptone. Additional literature data for CDOM in boreal forest soilporewaters (Wickland et al., 2007), soil amendments (Hunt and Ohno, 2007) and suldic to freshwater aquifer well and spring waters (Birdwell and Engel, 2009) are alsoprovided. The region of the plot to the left of the dashed line at FI 1.4 indicates terrestrially dominated CDOM; the region to the right of the dashed line at FI 1.9 indicates

olveresp

J.E. Birdwell, A.S. Engel / Organic Geochemistry 41 (2010) 270280 277microbially dominated CDOM; the region between the two lines represents unresindicate isolated aquatic humic substances of terrestrial and microbial provenance,where HIXZ is the Zsolnay HIX (dened previously) and HIXO is themodied version of the Zsolnay HIX described by Ohno (2002). FIand BIX values for the corn and dairy manure CDOM samples taken

BIX (autoc0 1 2

Hum

ifica

tion

Inde

x

0

10

20

30

40

50

60Suwannee River

fulvic acid

Pony Lakefulvic acid

Fig. 5. Comparison plot of BIX and HIX values for different CDOM types. Includes caveamendments (Hunt and Ohno, 2007) and aquifer well and spring waters (Birdwell and Eallochthonous CDOM; the region to the right of the dashed line at BIX 1.0 indicates autoCDOM signatures. SRFA and PLFA are indicated for reference.d (mixed) CDOM signatures. The locations of Suwannee River and Pony Lake FAsectively.from Hunt and Ohno (2007) were estimated from EEM contourplots reported in that study. FI and HIX values were reported by Bir-dwell and Engel (2009) for Edwards Aquifer well and spring water

hthonous index)3 4 5

Coal derived humicsSoil derived humicsAquatic humicsTryptoneSediment OMSediment PorewaterSoil Amendments(Hunt and Ohno, 2007)Cave and Spring watersMat ExtractsAquifer well and spring waters(Birdwell and Engel, 2009)

and spring waters and mat extracts, reference CDOM and literature data for soilngel, 2009). The region of the plot to the left of the dashed line at BIX 0.6 indicateschthonous CDOM; the region between the two lines represents unresolved (mixed)

ef

n w

ate)

pho

forand Pacic oceans from 200 miles from shore (Murphy et al., 2008), in the Balticradie inEl Trope

Geosamples, and BIX, Se, and a(375) values were calculated from those

Absorption Co1.0

Spec

tral S

lope

, Se (

nm-1

)

0.001

0.01Coal derived humicsSoil derived humicsAquatic humicsAtlantic and Pacific Ocea(Murphy et al., 2008)Baltic Sea waters(Kowalczuk et al., 2005) Danish Coastal waters(Stedmon et al., 2000)Sediment OMSediment PorewaterCave and Spring watersMat ExtractsAquifer well and spring w(Birdwell and Engel, 2009

Surface watersA

Fig. 6. Comparison plot of absorbance coefcient at 375 nm and spectral slope valuesreference CDOM. Also included are literature data for CDOM in waters of the AtlanticSea collected over a salinity gradient (Kowalczuk et al., 2005), on the Danish Coast gand in aquifer well and spring waters (Birdwell and Engel, 2009). The dashed linenvironments. SRFA and PLFA are indicated for reference and water samples from thepool (exposed to sunlight) to illustrate the effect of solar exposure on absorbance p

278 J.E. Birdwell, A.S. Engel / Organicpreviously reported uorescence and absorbance data. The range ofFI (1.101.25) and HIX (217) values reported by Huguet et al.(2009) placed their samples from the Gironde Estuary in the sameregion of Fig. 4 as the soil porewaters and soil amendments (e.g. Ta-ble 2), while comparing HIX and BIX (0.60.8) placed the estuarinesamples in a range similar to that of the sediment OM and pore-water samples (Table 2).

Although there is signicant scatter in the data (Fig. 4), HIX gen-erally decreases as FI increases up to ca. 1.8, at which point FI con-tinues to increase but HIX varies between 0.2 and ca. 5. The plot ofHIX and BIX shows a similar transition at HIX ca. 20 and BIX ca. 0.6(Fig. 5), which also provides an approximate cutoff between iso-lated humic substances, like the IHSS samples, and less renedsamples, such as the sediment extracts and porewaters (Table 2).FI and BIX provide similar ranges of values for the isolated humics(FI ca. 11.5; BIX ca. 0.30.7), but BIX had a somewhat wider rangeof values for the cave and spring waters (0.53.0) compared to FI(1.52.7).

The combination of HIX with either FI or BIX can be used toillustrate a continuum of CDOM uorescent characteristics (e.g. Ta-ble 2). Particular types of CDOM grouped more or less consistentlyin both comparisons. The continuum ranges between FI values ofca. 1 up to 1.8, with coal-derived HAs at one end (BIX ca. 0.35, FIca. 1, HIXP 50) and sediment-extracted and porewater CDOM atthe other (BIX ca. 0.8, FI 1.51.8, HIX ca. 10). Beyond FI ca. 1.8 orBIX ca. 0.8, CDOM in a particular sample is dominated by microbi-ally-derived substances that appear to have undergone little humi-cation (Fig. 5). The majority (60%) of the CDOM samples from thecave and spring waters falls within this range (Table 1), with therest lying between the terrestrially and microbially dominated re-gions of either gure (Figs. 4 and 5). Previously, these values wereset on the basis of criteria for microbially-derived CDOM(McKnight et al., 2001; Huguet et al., 2009), though it is still un-clear what the values of FI > 1.9 or BIX > 1 actually signify, becausesuch values have not been evaluated in detail. However, based on

ent from estuarine, brackish and near-oceanic environments (Stedmon et al., 2000)dicates the division between surface waters (inuenced by sunlight) and aphoticatio Geyser pool (Chile) collected near the spring discharge (aphotic) and the surfacerties.ficient = 375 nm (m-1)011

aters

rs

Suwannee Riverfulvic acid

Pony Lakefulvic acid

El TatioSurface Pool

El Tatio Springdischarge

tic waters

different CDOM types. Includes cave and spring waters and mat extracts along withchemistry 41 (2010) 270280the data collected for tryptone, which had an extremely low HIXvalue (0.05) and high BIX (5.16) and FI (2.45) values, it is possiblethat limited humication or structural differences in microbially-derived proteins and peptides may account for the range of highBIX and FI values observed for waters in this study.

4.3. Absorbance spectroscopy

Spectral slopes determined for the cave and spring sampleswere similar to those for aquifer waters, but were an order of mag-nitude lower than those for waters from Atlantic or Pacic Oceans(Murphy et al., 2008), the Baltic Sea (Kowalczuk et al., 2005), or theDanish Coast (Stedmon et al., 2000) (Fig. 6). The slopes were alsolower than those for the reference humic substances and DOMfrom surface waters and sediment porewaters. Values of the absor-bance coefcient a(375) were consistent with the range for litera-ture and reference CDOM samples. Although the spectral slopevalues distinguish the CDOM in the cave and spring waters fromCDOM found in other environments, the meaning of the differenceis uncertain. Studies of CDOM exposed to solar radiation report anincrease in spectral slope with duration of exposure (Grzybowski,2000; Vhtalo andWetzel, 2004). In contrast, microbial utilizationof DOM may atten the absorbance spectra of CDOM (Brown,1977; Stedmon et al., 2000). The lack of CDOM photodegradationin the cave and spring waters, along with signicant microbialactivity, may explain the unusually low spectral slope values.

5. Conclusions

The cave and spring waters displayed strong microbial uores-cence features, lacked signicant terrestrial signatures and exhib-ited unique absorbance characteristics. One of the mostimportant ndings is that a substantial portion of CDOM in karst

implies that either terrestrial CDOM does not contribute to the

uorescence indices can be used to describe a continuum of CDOM.

GeoDistinct CDOM groupings can be identied from BIX, FI and HIXvalues, as described in Table 2. The distinctions derived fromabsorbance spectral slopes are less clear for surface waters, but itis apparent that CDOM in cave and spring waters has differentUVVis absorbance characteristics than CDOM from other, primar-ily photic, environments. These spectroscopic differences are likelydue to a combination of environmental factors, including exposureto solar radiation, availability of organic carbon in the ecosystem oforigin, in situ microbial activity, and mixing of autochthonous andallochthonous CDOM. However, further work needs to be done todifferentiate among these various mechanisms.

Acknowledgements

The authors thank I. Warner, M. Lowry and R. Cook for equip-ment access and C. Schulz and K. Brannen for assistance with sam-ple collection, processing and analysis. We thank two anonymousreviewers for helpful suggestions. The work was partially fundedby the National Science Foundation (Chilean samples, EAR-0544960) and Louisiana Board of Regents Support Fund Grants(contract NSF/LEQSF (2005)-Pfund-04) for the Italian cave andother hot spring samples.

Associate EditorE. A. Canuel

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Characterization of dissolved organic matter in cave and spring waters using UVVis absorbance and fluorescence spectroscopyIntroductionBackgroundFluorescence spectroscopyFluorescence-derived indicesAbsorbance spectroscopy

Materials and methodsSample acquisition and geochemical characterizationAbsorbance and fluorescence measurements

Results and discussionGeneral spectral featuresFluorescence indicesAbsorbance spectroscopy

ConclusionsAcknowledgementsReferences