Characterization of dissolved organic matter in cave and spring waters using UV–Vis absorbance and fluorescence spectroscopy

Organic Geochemistry 41 (2010) 270–280

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Characterization of dissolved organic matter in cave and spring waters using UV–Vis absorbance and fluorescence spectroscopy Justin E. Birdwell a,*, Annette Summers Engel b a b

Cain Department of Chemical Engineering, Louisiana State University, Baton Rouge, LA 70803, USA Department of Geology and Geophysics, Louisiana State University, Baton Rouge, LA 70803, USA

a r t i c l e

i n f o

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

a b s t r a c t Chromophoric dissolved organic matter (CDOM) was examined using fluorescence and absorbance spectra from sulfidic cave and thermal and non-thermal surface-discharging spring waters. Many of the sites have a limited allochthonous supply of organic matter (OM) and contain ecosystems that are dependent on chemolithoautotrophic microbial communities. Water-extracted OM from microbial mats at the sites had fluorescence signatures consistent with the fluorescent amino acids. Based on fluorescence-derived indices and absorbance spectral characteristics, the origin of the cave and spring CDOM appeared to be from microbially-derived material, and the degree of OM humification was low. Little of the CDOM pool was represented by terrestrial humic fluorescence signatures, which are typically observed in surface waters, as well as soil and sediment porewaters. Comparison of the cave and spring waters with a wide array of reference humic substances and OM from other environments showed a continuum of spectral properties constrained by origin and degree of humification. Published by Elsevier Ltd.

1. Introduction Dissolved organic matter (DOM) has been intensely investigated in water systems around the world because of the significant roles this ubiquitous material plays in various biogeochemical and ecological processes (Findlay and Sinsabaugh, 2003; Anesio et al., 2004; Judd et al., 2006). In natural settings, DOM parent material is primarily derived from terrigenous and aquatic macro (e.g. plants, animals) and microorganisms (e.g. algae, phytoplankton and bacteria). Chromophoric dissolved organic matter (CDOM), the fraction that absorbs ultraviolet (UV) and visible light, is the controlling factor for the optical properties of surface waters (Green and Blough, 1994). Spectroscopic techniques can provide information about the source and composition of the DOM present in a system at natural abundance concentration, thereby eliminating the need for isolating or concentrating it prior to analysis (Coble, 1996; Hudson et al., 2007). There are notable differences in DOM composition between surface water and groundwater. The dominant components of DOM in most surface water environments are dissolved humic substances, primarily fulvic acids (Thurman, 1985). Relative to other types of DOM, humic substances accumulate in surface water environments because of their refractory nature (Frimmel, 1998). In contrast, ca. 30% of the DOM in uncontaminated subsurface waters consists of hydrophilic or neutral material not considered to be * Corresponding author. Tel.: +1 225 578 1426; fax: +1 225 578 1476. E-mail addresses: [email protected], [email protected] (J.E. Birdwell). 0146-6380/$ - see front matter Published by Elsevier Ltd. doi:10.1016/j.orggeochem.2009.11.002

recalcitrant, including polysaccharides, alkyl alcohols, aldehydes, ketones and amides (Leenheer, 1981). Based on research from groundwater (Leenheer and Noyes, 1984; Baker and Lamont-Black, 2001) and cave drip waters (e.g. Baker and Genty, 1999), the fluorescent amino acids generally attributed to microbial activity (tryptophan and tyrosine) account for most of the non-humic CDOM. Several processes may control the lack of surface-derived (terrigenous) humic substances in subsurface waters, including retention of terrestrial DOM by the soil column as water percolates into the subsurface, biotic molecular transformation of terrigenous DOM input (Einsiedl et al., 2007), or in situ DOM production by microbes indigenous to subsurface environments. Reactive mineral surfaces and energetically rich waters support an array of chemolithoautotrophic microorganisms in subsurface habitats (e.g. Stevens, 1997; Kinkle and Kane, 2000). When the allochthonous carbon input is limited or lacking altogether, these microbes can provide a source of organic carbon from inorganic carbon sources (e.g. bicarbonate) by gaining cellular energy from oxidation/reduction reactions, such as oxidation of reduced sulfur compounds (i.e. sulfur oxidation) or methanogenesis. Most terrestrial, chemolithoautotrophically-based ecosystems are associated with cave and karst terrains, whereby autochthonous OM is sufficient to support entire ecosystems. Some notable systems include the Movile Cave, Romania (Sarbu et al., 1996), the Frasassi Cave system in Central Italy (Vlasceanu et al., 2000) and Lower Kane Cave in Wyoming, USA (Engel et al., 2004). The CDOM properties of these cave waters are expected to be different from those of oceans, estuaries and freshwater near the

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surface, or soil and sediment porewaters, because of their unique biogeochemical conditions and lack of exposure to sunlight. However, our current understanding of microbially-derived DOM in subsurface ecosystems, which could account for a significant fraction of the DOM in the subsurface (e.g. Whitman et al., 1998), is incomplete because few studies, apart from those of material in the open ocean (e.g. Yamashita and Tanoue, 2003), have been conducted on the CDOM from systems that are not significantly influenced by terrigenous DOM sources or photosynthetically-driven microbial activity. Photosynthetically-driven microbial activity has been shown to produce humic-like substances in the absence of 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 Fryxell in Antarctica (e.g. Aiken et al., 1996; McKnight et al., 2001; Fulton et al., 2004). In this study, the UV–Vis spectroscopic properties of CDOM from a variety of cave and spring waters were investigated, including sulfidic karst springs and several geothermal, non-karst springs. The sites are important microbial habitats (e.g. Sarbu et al., 1996; Vlasceanu et al., 2000; Engel et al., 2004, 2008; Porter and Engel, 2008). Our goal was to determine how the fluorescence and absorbance characteristics of water and microbiological material from these systems compare with each other and with CDOM collected from surface environments, including isolated OM and humic substances. Our assessment of these waters provides a distinctive and novel array of CDOM signatures constrained by microbial influences that can serve as a comparison for future water resource investigations and carbon budget studies. From a management and conservation perspective, knowledge of CDOM characteristics and sources is critical for preserving groundwater quality and ecosystem integrity, especially for systems that may be highly susceptible to contamination, like karst aquifers that supply a significant portion of the world’s drinking water (Spizzico et al., 2005; Green et al., 2006; Birdwell and Engel, 2009).

2001; Huguet et al., 2009) and various stages of humification or diagenesis (Zsolnay et al., 1999). There are a number of different methods for interpreting fluorescence data, from peak picking to complex numerical modeling schemes like Parallel Factor Analysis (e.g. Coble, 1996, 2007; Stedmon et al., 2003; Stedmon and Markager, 2005; Cory and McKnight, 2005; Hunt and Ohno, 2007; Murphy et al., 2008; Cook et al., 2009). Excitation–emission matrix (EEM) fluorescence spectroscopy uses an assembly of fluorescence emission spectra collected over a range of excitation wavelength to summarize the entire steady-state UV and visible fluorescence behavior of a particular sample (Fig. 1). EEM spectra provide information on the relative intensity of fluorescence at different excitation–emission wavelength pairs (or regions) and the Stoke’s shifts of fluorophores in a way that is fast and convenient, particularly for complex mixtures of fluorescent components (Coble, 1996). The typical region of the UV and visible fluorescence spectrum obtained on DOM samples is from the mid-UV (or UVC, >200 nm) to the violet and near-blue (ca. 450 nm) in the excitation wavelength (kEx), and emission wavelengths (kEm) from mid-UV to the yellow or near-orange (ca. 600 nm). In general, little CDOM fluorescence emission is observed past kEm ca. 550 nm (Smith and Kramer, 1999). Coble (1996, 2007) identified a set of characteristic CDOM descriptive peaks in EEM fluorescence spectra. These peaks have been observed in spectra collected from filtered surface waters, as well as isolated humics and OM samples. There are three commonly observed humic-like peaks (Fig. 1): UVC-excited (designated A; kEx 240–260 nm, kEm 400–460 nm), UVA-excited (designated C; kEx 320–360 nm, kEm 420–460 nm) and marine humics (designated M, kEx 290–310 nm, kEm 370–410 nm), though peak M has also been attributed to coastal and marine biological activity (Parlanti et al., 2000), anthropogenic input to natural waters (Stedmon and Markager, 2005) and has been proposed as a precursor for peak C fluorophores (Burdige et al., 2004). Peaks indicative of biological activity or protein-like material include tyrosine-like peaks (B, kEx 270–280 nm, kEm 300–315 nm) and tryptophan-like peaks (T, kEx 270–280 nm, kEm 345–360 nm).

2. Background

2.2. Fluorescence-derived indices

2.1. Fluorescence spectroscopy

Fluorescence intensity ratios can be used to infer the relative contributions from autochthonous and allochthonous OM in natural waters. McKnight et al. (2001) found that the ratio of the emission intensity at kEm 450 nm to that at kEm 500 nm, following excitation at kEx 370 nm, provides a metric for distinguishing CDOM derived from terrestrial and microbial sources. This ratio is referred to as the fluorescence index (FI; Fig. 1). FI has been calibrated using data collected on fulvic acids and whole water samples obtained from environments known to contain CDOM derived from either allochthonous or autochthonous sources (McKnight et al., 2001). FI values can be influenced primarily by the intensities of the UVA-excited humic peak (C), peak M, and possibly other, as yet unidentified, fluorophores with large (>80 nm) Stoke’s shifts that emit in the blue–green region of the visible spectrum (Burdige et al., 2004). FI values of 1.4 or less indicate DOM of terrestrial origin and values of 1.9 or higher correspond to microbially-derived material. An inverse relationship exists between FI and DOM aromaticity, 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 expected to contain more conjugated aromatic structures than microbially-derived substances and terrestrially-derived humic substances should have greater emission intensity at longer wavelength as a result of the increase in the C/H ratio during diagenesis (Stevenson, 1982). Consequently, the ratio of short (kEm 450 nm) to long wavelength (kEm 500 nm) emitting fluorophores

Fluorescence spectroscopy is a highly selective technique for analyzing organic substances because only those compounds containing moieties with conjugated bonds are observed (Lakowicz, 1999). The method is also highly sensitive at natural abundance levels and analyte concentration through isolation is generally not needed (Coble, 1996). There are two major categories of fluorophores in uncontaminated natural waters: humic-like and proteinlike (Baker and Lamont-Black, 2001; Chen et al., 2003). Humic-like fluorescence is a term used to describe spectral features that resemble those of isolated humic and fulvic acids (Alberts and Takács, 2004), while the term protein-like fluorescence describes peaks that are attributed to the fluorescent amino acids tryptophan and tyrosine (Yamashita and Tanoue, 2004). Both humic-like and protein-like fluorophores absorb, and are excited by, UV light (240–280 nm). Humic-like fluorophores emit primarily in the violet to near-green (400–500 nm), which results from the presence of quinone-like structures sourced from the degradation of terrestrial biomaterial such as lignin (Ariese et al., 2004; Cory and McKnight, 2005). In contrast, protein-like fluorophores emit in the UV to near-violet (300–380 nm). These starkly different humic-like and protein-like fluorescence characteristics allow the selective and sensitive distinction of the relative contributions of natural materials in DOM (e.g. Coble, 1996; Chen et al., 2003; Hudson et al., 2007) that possibly reflect different origins (e.g. McKnight et al.,

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450

400

ay

le

ig

h

Fluorescence Index (FI)

de

rR

350

or

st

Groundlevel Solar Radiation

300

Peak C

BIX

1

Excitation Wavelength (nm)

Suwannee River fulvic acid

Peak M

Humification Index (HIX)

Peak B Peak T

nd

2

Peak A

250 250

300

350

400

450

igh yle Ra r e ord

500

550

Emission Wavelength (nm) Fig. 1. Summary of CDOM characteristics of natural water samples. EEMs for three typical CDOM components in whole water samples are shown: dashed–dotted contour lines represent tyrosine, solid lines tryptophan, and dashed lines Suwannee River FA, a terrestrially-derived (or allochthonous) aquatic humic material. White ovals and circles represent the Coble (1996, 2007) peak designations A, C, M, T and B (see Section 2 for more information). Locations of emission intensities used to calculate HIX (thick solid lines, kEx 254 nm), BIX (dark gray points, kEx 310 nm) and FI (light gray points, kEx 370 nm) are indicated. Additional lines indicate position of 1st and 2nd order Rayleigh scattering bands (solid) and position where ground level solar radiation intensity becomes important for photochemical processes (k > ca. 300 nm).

in microbially-derived OM is expected to be higher than for OM from terrestrial sources. Another index was proposed recently to assess the relative contribution of autochthonous DOM in water samples, called the biological/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 fixed excitation (kEx 310 nm; Fig. 1). Formulation of BIX was influenced by the presence of two common peaks in fluorescence spectra collected on surface waters, attributed to terrestrial and microbial components. 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). The longer emission wavelength peak is greatly affected by the nearby UVA-excited terrestrial humic peak C, which is considered an indicator of allochthonous carbon in aquatic systems (Coble, 1996, 2007) or a more diagenetically altered form of peak M fluorophores (Burdige et al., 2004). Values of BIX between 0.8 and 1.0 correspond to freshly produced DOM of biological or microbial origin, whereas values below ca. 0.6 are considered to contain little autochthonous OM. The degree of DOM humification is an indicator of a material’s age and recalcitrance within a natural system (Zsolnay et al., 1999; Ohno, 2002). Highly humified organic substances are generally resistant to degradation and are expected to persist in the environment longer than substances with a low degree of humification. Emission fluorescence has been used to estimate the degree of humification of DOM extracted from soils and other OM sources using various spectral analyses. Zsolnay et al. (1999) proposed a humification index (HIX) determined from the ratio of two integrated regions of an emission scan (sum from kEm 435–480 nm divided by the sum from kEm 300–345 nm) collected with excitation at 254 nm as a method for comparing the relative humification of DOM samples (Fig. 1). HIX is low (<5) for fresh DOM derived from plant biomass and animal manure (Ohno and Bro, 2006; Hunt and

Ohno, 2007; Ohno et al., 2007) and generally increases with degree of decomposition (Hunt and Ohno, 2007; Wickland et al., 2007) or fractionation of DOM by sorption onto mineral surfaces (Ohno et al., 2007). Water extractable DOM from soil and soil porewater has HIX values between 10 and 30 (Kalbitz et al., 2003; Wickland et al., 2007). Huguet et al. (2009) applied the HIX to estuarine samples across a salinity gradient and found that it generally decreased with increasing salinity (values ranged from 2 to 17). HIX is strongly correlated with DOM aromaticity and inversely correlated with carbohydrate content (Kalbitz et al., 2003). 2.3. Absorbance spectroscopy Absorption of UV and visible light by CDOM generally decreases exponentially with increasing wavelength. This has been modeled in a number of studies (Stedmon et al., 2000; Kowalczuk et al., 2005; Murphy et al., 2008), using the following equation

aðkÞ ¼ aðkref Þ  eSe ðkkref Þ þ K

ð1Þ 1

where a(k) is the absorption coefficient at wavelength k (m ), kref the reference wavelength (nm), Se the spectral slope parameter (nm1) and K, a background correction parameter to account for baseline shift due to scattering (m1) (Stedmon et al., 2000; Kowalczuk et al., 2005); a(kref) is proportional to dissolved carbon concentration in waters containing similar types of chromophores (Stedmon et al., 2000). Se has been related to the ratio of fulvic to humic 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 sulfidic, non-sulfidic (freshwater)

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Table 1 Description of water and microbial mat samples and summary of fluorescence maximum and total intensity, absorbance characteristics and fluorescence and humification indices.

a b c d e

Sampling site (Fig. 2 panel)

Temp (°C)

pH

TDS (mg l1)

IMax (RU)

kEm Max (nm)

kEx Max (nm)

FI

HIX

BIX

a(375) (m1)

Se  1000 (nm1)

Baker Hot Springs, WA, USAa (a) Big Sulphur Cave, KY, USAa,b,c (b)

36.2 15

8.2 7.2

548.6 NM

1.49 4.76

447.5 415

240 240

1.63 1.91

2.06 1.21

0.51 0.48

1.65 1.89

0.94 1.48

Frasassi Caves, Italy Resurgence Springa,c Ramo Sulfureoa,b,c Sulfide surface wella,b,c (g) Grotta Bellaa,b,c (h)

13.8 13.6 13.7 13.8

7.4 7.1 7.4 7.3

1408 1350 1396 1402

3.79 2.71 2.32 2.16

357.5 412.5 410 420

240 240 240 240

1.96 1.80 1.80 1.81

0.28 1.79 3.28 1.91

2.50 1.12 0.93 0.80

1.54 1.55 1.55 1.51

0.44 0.59 0.60 0.65

Mat waters Pozzo di Cristalia,b,c (m) Sulfide surface wella,b,c (n) Grotta Bellaa,b,c (o)

13.4 13.7 13.8

7.2 7.4 7.3

1903 1396 1402

3.08 3.15 2.72

317.5 300 400

240 270 240

1.99 1.62 2.29

0.25 0.29 0.40

1.52 1.95 0.99

2.83 2.81 2.79

1.92 2.03 1.39

Glenwood Hot Spring, CO, USAa,b,c (c) Jemez Spring, NM, USAa

48 46.6

6.4 5.9

18,744 5264

1.13 2.04

407.5 415

290 240

1.97 2.10

0.62 1.21

0.78 0.86

1.50 2.88

0.42 1.49

Lower Kane Cave, WY, USA Mat water (200 m)a,b,c (l) Water (63 m)a,b,c (e) Water (200 m)a,b,c

22.5 22.5 22.5

7.5 7.4 7.5

393 388 393

4.48 2.52 2.00

352.5 352.5 307.5

240 240 240

2.00 2.30 1.78

0.26 0.22 0.14

2.36 1.30 NDe

ND 2.14 2.18

ND 1.58 1.66

Pah Tempe Hot Springs, UT, USA Mat watera (k) Alcove watera

44–5 44–5

6.9 6.9

NMd NM

8.08 1.47

365 310

270 240

1.63 1.74

0.22 0.12

1.26 ND

2.24 2.21

1.59 1.56

Sharon Springs, NY, USAa (d) Sulfur Springs, IN, USAa,c

10 13.7

6–7 7.3

NM 3876

7.16 1.85

347.5 315

275 240

1.92 2.06

0.61 0.36

0.64 1.13

2.63 3.00

2.04 1.61

El Tatio ‘‘Main Geyser”, Chile Mat water (j) Surface water

59 77

7.1 6.8

12,890 11,952

5.92 1.02

355 280

300 250

2.63 1.91

1.36 0.52

4.92 0.65

2.20 8.50

1.54 4.56

Terme di Agnano spring, Italya (i) White Sulphur Springs, LA, USAa (f)

56.7 18.7

6.4 6.8

1973 NM

1.73 2.32

300 307.5

270 275

2.00 2.28

1.16 0.86

0.92 0.85

2.76 3.13

1.52 1.71

Sulfidic. Aphotic. Karst. NM, not measured. ND, not determined.

and geothermal systems (Table 1). Some of the spring waters that were exposed to sunlight upon reaching the surface were collected near the source to limit the effect of solar irradiation. Each water sample was passed through a 0.45 lm Whatman glass fiber filter (GF/F, autoclaved and precombusted at 500 °C) and a 0.2 lm PTFE (Millipore, Bedford, MA) filter in tandem before storage in HDPE bottles. For some sites, raw filamentous microbial mats were collected with site water. Following centrifugation and filtration, the mat waters (herein referred to as mat extracts) were diluted by 100–1000-fold to obtain solutions with UV absorbance (240 nm) of ca. 0.1 and to be consistent with the water samples. Filtered water and mat extracts were stored on ice for transport and maintained at 4 °C until analysis. Depending on the circumstances of collection for each water sample, storage times varied between <1 week and up to 4 years. We conducted internal tests to determine if storage time affected the fluorescence and absorbance properties of the samples. Several of the samples collected during the last two years of the study were run multiple times following their collection. There were no discernible changes observed in the character of the fluorescence and absorbance spectra and there were no changes in peak intensities greater than 2%, within the manufacturer’s suggested tolerance. Unstable geochemical parameters were measured immediately in the field using standard electrode methods (e.g. American Public Health Association (APHA), 1998), including temperature and pH with a double junction electrode (Accumet AP62 meter, Fisher Scientific, USA), and total dissolved solids (TDS) and temperature (YSI-85 meter, Yellow Springs, OH, USA).

3.2. Absorbance and fluorescence measurements All fluorescence measurements were made using a SPEX Fluorolog-3 spectrofluorometer (Jobin Yvon, Edison, NJ, USA) equipped with a 450 W Xe lamp, double excitation and emission monochromators and an extended red, high sensitivity, multi-alkali photocathode photomultiplier tube (Hamamatsu Corporation, Bridgewater, NJ, USA) with slits set to 5 nm for both excitation and emission monochromators and using a 0.1 s integration time. A 1 cm quartz cuvette was used at room temperature (ca. 22 °C). Instrument stability was determined using the Raman peak of deionized water excited at 348 nm, with emission monitored at 395 nm. Water Raman intensities were consistent during each session, with values varying <2% between runs, in agreement with specifications from the manufacturer. Samples were analyzed in signal/reference mode, where the fluorescence emission intensity is normalized to the intensity of the lamp at the particular excitation wavelength applied. Spectra were not adjusted using the manufacturer-provided instrument correction factors and are therefore comparable to those of McKnight et al. (2001). EEM fluorescence spectra were obtained by collecting a series of 43 emission scans (kEm 250–550 nm, 2.5 nm intervals) at 5 nm excitation wavelength intervals between kEx 240 and 450 nm. EEM plots were assembled by combining the individual emission spectra using SigmaPlot 10 (Systat Software, Inc., San Jose, CA, USA) and contain 10 contour lines, with each line representing 10% of the maximum emission intensity. Fluorescence emission intensities are reported in Raman Units (RU), where the sample

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intensities are normalized to the emission intensity of the deionized water Raman signal at kEx 348 nm, kEm 395 nm. UV–Vis absorbance spectra were collected using a double beam UV-3101PC spectrophotometer (Shimadzu Corporation, Kyoto, Japan) and a 1 cm quartz cuvette over the range 200–600 nm with deionized water as the reference. To fit the data to Eq. (1), optical density (absorbance value) was converted to absorption coefficient:

aðkÞ ¼

2:303AðkÞ l

ð2Þ

where A(k) is the optical density at wavelength k (dimensionless) and l the length of the cell used in the absorbance measurement (m). Eq. (1) was fitted to absorbance data between 300 and 600 nm, and 375 nm was the reference wavelength. The absorbance data were fitted using a non-linear regression technique, rather than the linearized form of Eq. (1), because the non-linear approach has been found to yield smaller residuals (Kowalczuk et al., 2005). Spectral corrections for primary and secondary inner filter effects in the fluorescence spectra were made using absorbance data (Lakowicz, 1999). Raman scattering was mitigated by subtracting a blank EEM spectrum collected on pyrogen-free deionized (>18.1 MX) water from each corrected EEM. Rayleigh scattering effects were edited from each spectrum following correction and blank subtraction. FI, BIX, and HIX values were determined using data from the corrected EEM spectra. A set of reference fluorescence spectra was obtained to represent CDOM from different surface environments, and compounds whose fluorescence characteristics are similar to CDOM spectral features observed in other studies. Samples from the International Humic Substances Society (IHSS) served as proxies for dissolved humic substances (humic and fulvic acids, HAs and FAs, respectively) derived from soil (Elliot HA, Pahokee HA, Wakish HA and FA), surface waters (Nordic HA, FA and NOM; Suwannee River HA, FA and NOM; Pony Lake FA) and coal (Leonardite HA). Additional humic isolates included Amherst HA (soil), Laurentian HA and FA (soil), Aldrich HA (coal) and a series of sediment humic fractions extracted using 0.1 N NaOH from six sites in Louisiana, New York and Maryland. Porewater was extracted from the sediment samples by centrifugation and analyzed following filtration (0.45 lm GF/F, 0.2 lm PTFE). The reference humics and sediment derived samples were used in a recent study of DOM isolated from the Atchafalaya Basin (Cook et al., 2009). Other standards included L() and D(+) tryptophan (Acros Organics, Thermo Fisher Scientific, Inc., NJ, USA), L() and D() tyrosine (Acros Organics) and tryptone (Fisher Bioreagents, Fisher Scientific, Inc., NJ, USA) to obtain a protein-like signature. All comparison samples were made or diluted using deionized water and prepared such that the concentrations were ca. 10 mg organic carbon l1. The final pH of the reference materials 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 a through i) contained many of the characteristic peaks observed in other studies of marine and terrestrial CDOM (e.g. Fig. 1). The peaks attributed to fluorescent amino acids were most evident in the spectra collected from the mat extracts (Fig. 2, panels j–o). The spectra for the IHSS reference collection and other terrestrial and aquatic samples (data not shown) were composed primarily of the UVC and UVA-excited humic peaks (A and C), as described by Coble (1996). The maximum emission intensities observed for Suwannee 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 range of wavelengths investigated) and their position differed by ca. 15 nm (437.5 and 422.5 nm, respectively). The typical emission maxima for the cave and spring water samples were at the short end of the wavelength range for peak A fluorophores (kEm ca. 400–420 nm). The dominant protein-like peaks from mat extracts were attributed to tyrosine (kEm ca. 300 nm) and tryptophan (kEm ca. 350 nm), corresponding to the peak B and T regions, respectively. The tryptone spectrum was dominated by tryptophan fluorescence (peak T) and also contained a less intense tyrosine-like peak (B). Corrected maximum fluorescence emission intensity (IMax) for all waters from the caves and springs sampled was between 1 and 10 RU. The absorbance spectra for the cave and spring waters had steep drops in optical density between 200 and 250 nm, followed by an exponential decrease with increasing wavelength beyond 280 nm. Many samples had a peak or shoulder in the ca. 260– 270 nm range, consistent with strong absorbance by fluorescent amino acids, but could also be due to the presence of a wide range of other specific compounds that absorb in this region of the UV spectrum. Absorbance coefficients determined at 375 nm ranged from 1.50 (Glenwood Hot Springs, CO) to 8.50 m1 (El Tatio Geyser Pool, Chile), with an average of 2.52 m1 (standard deviation 1.51 m1). Spectral slopes determined using Eq. (1) for the waters varied from 4.20  104 (Glenwood Hot Springs, CO, USA) to 4.56  103 nm1 (El Tatio Geyser Pool), with an average slope for all samples of 1.48  103 nm1 (std. dev. 8.95  104) and coefficient of determination (R2) of P0.90. The humic and CDOM reference materials had a(375) values between 0.71 (NY harbor sediment porewater) and 12.27 m1 (Elliot soil HA, IHSS), and spectral slopes between 7.30  103 (Pahokee Peat HA, IHSS) and 1.68  102 nm1 (LA salt marsh sediment porewater), with R2 P 0.98. Fig. 3 contains a set of reference UV–Vis absorbance spectra for various humic standards, as well as the spectrum collected for a sample from the Frasassi Cave system in Italy (Ramo Surfureo). 4.2. Fluorescence indices None of the water or mat extracts had FI values <1.6 (Table 1). FI values were >1.9 for half of the samples, with three samples having values > 2.2. These higher values are similar to those of filtered whole water samples collected from a perennially ice-covered Antarctic lake (McKnight et al., 2001 and references therein) and the samples with FI values between 1.6 and 1.9 are consistent with significant quantities of microbially-derived CDOM (e.g. McKnight et al., 2001), but the relative contribution of autochthonous and allochthonous material cannot be discerned from the FI values. All the coal, soil-derived and aquatic reference humic substances had 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). DOM extracted from sediment particles or present in sediment porewaters had values of ca. 1.75, consistent with material representing a mixture of allochthonous and autochthonous sources. Tryptone had a value of 2.45, higher than all but one mat extract (El Tatio Main 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 Sulfur Cave (KY, USA), which had values between ca. 0.5 and 0.7. The mat samples had values of ca. 1 or higher. These high values for the cave and spring water samples are consistent with those observed by Huguet et al. (2009) for autochthonous DOM derived from microbially-dominated samples. The reference humic substances from coal, soil and surface waters demonstrated a wider range of

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Cave and Spring Waters 450

450

Excitation Wavelength (nm)

a

450

b

c

400

400

400

350

350

350

300

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Emission Wavelength (nm) Fig. 2. Representative EEMs for cave and spring filtered water samples (a–i) and EEMs for mat extracts (j–o) from locations with accessible microbial mats (see Table 1 for sample information and correspondence with panel letter).

BIX than FI values (0.31–0.78), though >80% had BIX values <0.4. The exceptions were Laurentian HA (0.78), Pahokee Peat HA (0.63) and Pony Lake FA (0.66). Sediment-extracted and porewater DOM had BIX values between 0.57 (LA salt marsh porewater) and 1.21 (LA lake sediment-OM) and 75% of the samples had values >0.7. BIX for tryptone was higher (5.16) than all of the water sam-

ples, although the highest value among the mat extracts was similar (El Tatio Main Geyser; BIX 4.92). All the cave and spring waters had HIX values <3.3 (Table 1). Only two had values >2 (Baker Hot Springs, WA; Sulfide Surface Well, Frasassi Caves, Italy). Such low values are consistent with the majority of the CDOM being principally composed of fresh

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-1

Absorbance (cm )

1

0.1 Leonardite HA Ramo Sulfureo Suwannee River FA 0.01

Pony Lake FA

0.001 200

300

400

500

600

Wavelength (nm) Fig. 3. UV–Vis absorbance spectra comparing terrestrial (Suwannee River FA and Leonardite HA) and microbial (Pony Lake FA) humic substances to a representative water sample from the Frasassi Cave system in Italy (Ramo Sulfureo).

biological detritus, similar to material derived from lysed cells obtained by fumigating soil samples with CHCl3 (Zsolnay et al., 1999) and fresh organic substances extracted from plant biomass and animal manure (Ohno and Bro, 2006; Hunt and Ohno, 2007; Ohno et al., 2007). The reference humics had much higher HIX values than the cave and spring samples (HIX ca. 10–60), consistent with the materials representing isolated, recalcitrant fractions of DOM from coal, soil and surface waters. The HIX values of the coal derived humics were the highest (>50). Soil and aquatic humics had a wide range of values (10–30 and 20–50, respectively) and the sediment-derived and porewater DOM samples were somewhat less humified (HIX ca. 6–20), with the exception of one sample (LA salt marsh porewater; HIX 28.37). Tryptone had a low value (0.05), which may or may not be meaningful, although it appears to represent a wholly unhumified biological signature. Table 2 provides a summary of the range of HIX, FI and BIX values for particular types of CDOM examined in this study. For the water samples, the low HIX and high BIX and FI values may represent fluorescent, water soluble, extracellular substances excreted by microorganisms, detritus resulting from cell death, and aquatic humic substances less humified than surface water humics, possibly due to a lack of exposure to solar radiation. Specifically, pro-

duction of humified CDOM signatures by microbial processing has been shown to be reduced in the absence of sunlight in simulated estuarine systems (Stedmon and Markager, 2005). CDOM in these cave and spring waters could be an active constituent of the local carbon cycle at our study sites. These findings support earlier assertions that microbial DOM processing in caves is an important process (e.g. Simon et al., 2007) and agrees with findings that suggest rapid, microbially-driven DOM transformation in karst aquifers occurring of the order of decades (Einsiedl et al., 2007). An inverse relationship between FI and HIX, implied by their correlation with DOM aromaticity (McKnight et al., 2001; Kalbitz et al., 2003), was evaluated with data from all the samples and reference standards, and values in the literature (Wickland et al., 2007; Hunt and Ohno, 2007; Huguet et al., 2009; Birdwell and Engel, 2009; Fig. 4). The HIX values reported by Wickland et al. (2007) were calculated using the method suggested by Ohno (2002) and were converted to values consistent with the HIX of Zsolnay et al. (1999) using the following relationship developed from a simple algebraic transformation

HIXZ ¼

1 1=HIXO  1

ð3Þ

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

a b

Category (Ref.)

FI range

HIX range

BIX range

Isolation or collection

Coal derived humics Soil derived humics Sediment OM Aquatic humics Soil porewater (Wickland et al., 2007) Sediment porewater Soil amendments (Hunt and Ohno, 2007) Cave and spring waters (this study) Microbial mats, aqueous extracts (this study) Protein (tryptone) (this study)

1.0–1.2 0.8–1.3 1.2–1.6 1.0–1.3a 1.2–1.5 1.5–1.8 1.2–1.4 >1.6 >2 >2 (2.45)

>50 10–30 5–20 20–50 5–25 5–30 <5 <5 <2 <1 (0.05)

0.35 0.3–0.78 0.58–1.22 0.30–0.40a NDb 0.57–1.10 0.45 0.5–3.0 >2.0 2.0 (5.16)

IHSS Leonardite and Aldrich HAs Humic and fulvic acids from the IHSS and Amherst and Laurentian humics Base-extracted, unfractionated humic substances HAs and FAs from the IHSS Filtered whole water Extracted by centrifugation and filtered Water extracted solution from fresh plant biomass and animal manure Filtered whole water Extracted by centrifugation and filtered Casein digest

Pony Lake FA, the microbially-derived reference material, had FI 1.51 and BIX 0.66. ND, not determined.

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Coal derived humics Soil derived humics Aquatic humics Tryptone Sediment OM Sediment Porewater Soil Porewater (Wickland et al., 2007) Soil Amendments (Hunt and Ohno, 2007) Cave and Spring waters Mat Extracts Aquifer well and spring waters (Birdwell and Engel, 2009)

60 Suwannee River fulvic acid

Humification Index

50

40

30

Pony Lake fulvic acid

20

10

0 1.0

1.5

2.0

2.5

Fluorescence Index 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 soil porewaters (Wickland et al., 2007), soil amendments (Hunt and Ohno, 2007) and sulfidic to freshwater aquifer well and spring waters (Birdwell and Engel, 2009) are also provided. 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 microbially dominated CDOM; the region between the two lines represents unresolved (mixed) CDOM signatures. The locations of Suwannee River and Pony Lake FAs indicate isolated aquatic humic substances of terrestrial and microbial provenance, respectively.

where HIXZ is the Zsolnay HIX (defined previously) and HIXO is the modified version of the Zsolnay HIX described by Ohno (2002). FI and BIX values for the corn and dairy manure CDOM samples taken

from Hunt and Ohno (2007) were estimated from EEM contour plots reported in that study. FI and HIX values were reported by Birdwell and Engel (2009) for Edwards Aquifer well and spring water

Coal derived humics Soil derived humics Aquatic humics Tryptone Sediment OM Sediment Porewater Soil Amendments (Hunt and Ohno, 2007) Cave and Spring waters Mat Extracts Aquifer well and spring waters (Birdwell and Engel, 2009)

60 Suwannee River fulvic acid

Humification Index

50

40

30

Pony Lake fulvic acid

20

10

0 0

1

2

3

4

5

BIX (autochthonous index) Fig. 5. Comparison plot of BIX and HIX values for different CDOM types. Includes cave and spring waters and mat extracts, reference CDOM and literature data for soil amendments (Hunt and Ohno, 2007) and aquifer well and spring waters (Birdwell and Engel, 2009). The region of the plot to the left of the dashed line at BIX 0.6 indicates allochthonous CDOM; the region to the right of the dashed line at BIX 1.0 indicates autochthonous CDOM; the region between the two lines represents unresolved (mixed) CDOM signatures. SRFA and PLFA are indicated for reference.

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-1

Spectral Slope, Se (nm )

Surf ace

0.01

0.001

wate Suwannee River rs fulvic acid Coal derived humics Aph otic w Soil derived humics aters Aquatic humics Atlantic and Pacific Ocean waters Pony Lake (Murphy et al., 2008) fulvic acid El Tatio Baltic Sea waters Surface Pool (Kowalczuk et al., 2005) Danish Coastal waters (Stedmon et al., 2000) Sediment OM Sediment Porewater Cave and Spring waters Mat Extracts El Tatio Spring Aquifer well and spring waters discharge (Birdwell and Engel, 2009)

0.1

1

10 -1

Absorption Coefficient λ = 375 nm (m ) Fig. 6. Comparison plot of absorbance coefficient at 375 nm and spectral slope values for different CDOM types. Includes cave and spring waters and mat extracts along with reference CDOM. Also included are literature data for CDOM in waters of the Atlantic and Pacific oceans from <2 to >200 miles from shore (Murphy et al., 2008), in the Baltic Sea collected over a salinity gradient (Kowalczuk et al., 2005), on the Danish Coast gradient from estuarine, brackish and near-oceanic environments (Stedmon et al., 2000) and in aquifer well and spring waters (Birdwell and Engel, 2009). The dashed line indicates the division between surface waters (influenced by sunlight) and aphotic environments. SRFA and PLFA are indicated for reference and water samples from the El Tatio Geyser pool (Chile) collected near the spring discharge (aphotic) and the surface pool (exposed to sunlight) to illustrate the effect of solar exposure on absorbance properties.

samples, and BIX, Se, and a(375) values were calculated from those previously reported fluorescence and absorbance data. The range of FI (1.10–1.25) and HIX (2–17) values reported by Huguet et al. (2009) placed their samples from the Gironde Estuary in the same region of Fig. 4 as the soil porewaters and soil amendments (e.g. Table 2), while comparing HIX and BIX (0.6–0.8) placed the estuarine samples in a range similar to that of the sediment OM and porewater samples (Table 2). Although there is significant scatter in the data (Fig. 4), HIX generally decreases as FI increases up to ca. 1.8, at which point FI continues to increase but HIX varies between 0.2 and ca. 5. The plot of HIX and BIX shows a similar transition at HIX ca. 20 and BIX ca. 0.6 (Fig. 5), which also provides an approximate cutoff between isolated humic substances, like the IHSS samples, and less refined samples, such as the sediment extracts and porewaters (Table 2). FI and BIX provide similar ranges of values for the isolated humics (FI ca. 1–1.5; BIX ca. 0.3–0.7), but BIX had a somewhat wider range of values for the cave and spring waters (0.5–3.0) compared to FI (1.5–2.7). The combination of HIX with either FI or BIX can be used to illustrate a continuum of CDOM fluorescent characteristics (e.g. Table 2). Particular types of CDOM grouped more or less consistently in both comparisons. The continuum ranges between FI values of ca. 1 up to 1.8, with coal-derived HAs at one end (BIX ca. 0.35, FI ca. 1, HIX P 50) and sediment-extracted and porewater CDOM at the other (BIX ca. 0.8, FI 1.5–1.8, HIX ca. 10). Beyond FI ca. 1.8 or BIX ca. 0.8, CDOM in a particular sample is dominated by microbially-derived substances that appear to have undergone little humification (Fig. 5). The majority (60%) of the CDOM samples from the cave and spring waters falls within this range (Table 1), with the rest lying between the terrestrially and microbially dominated regions of either figure (Figs. 4 and 5). Previously, these values were set on the basis of criteria for microbially-derived CDOM (McKnight et al., 2001; Huguet et al., 2009), though it is still unclear what the values of FI > 1.9 or BIX > 1 actually signify, because

such values have not been evaluated in detail. However, based on the data collected for tryptone, which had an extremely low HIX value (0.05) and high BIX (5.16) and FI (2.45) values, it is possible that limited humification or structural differences in microbiallyderived proteins and peptides may account for the range of high BIX and FI values observed for waters in this study. 4.3. Absorbance spectroscopy Spectral slopes determined for the cave and spring samples were similar to those for aquifer waters, but were an order of magnitude lower than those for waters from Atlantic or Pacific Oceans (Murphy et al., 2008), the Baltic Sea (Kowalczuk et al., 2005), or the Danish Coast (Stedmon et al., 2000) (Fig. 6). The slopes were also lower than those for the reference humic substances and DOM from surface waters and sediment porewaters. Values of the absorbance coefficient a(375) were consistent with the range for literature and reference CDOM samples. Although the spectral slope values distinguish the CDOM in the cave and spring waters from CDOM found in other environments, the meaning of the difference is uncertain. Studies of CDOM exposed to solar radiation report an increase in spectral slope with duration of exposure (Grzybowski, 2000; Vähätalo and Wetzel, 2004). In contrast, microbial utilization of DOM may flatten the absorbance spectra of CDOM (Brown, 1977; Stedmon et al., 2000). The lack of CDOM photodegradation in the cave and spring waters, along with significant microbial activity, may explain the unusually low spectral slope values. 5. Conclusions The cave and spring waters displayed strong microbial fluorescence features, lacked significant terrestrial signatures and exhibited unique absorbance characteristics. One of the most important findings is that a substantial portion of CDOM in karst

J.E. Birdwell, A.S. Engel / Organic Geochemistry 41 (2010) 270–280

waters, and especially in sulfidic cave systems, appears to be almost exclusively a result of in situ microbial activity. The results are not unexpected given the prevalence of microorganisms and microbial mats in these systems. However, interestingly, other subsurface and spring waters were similar to the CDOM spectroscopic characteristics from the sulfidic spring sites. The fluorescence-derived indices provide support for a significant microbial role in the DOM sources and types in a variety of subsurface habitats, and indicate that unaltered allochthonous CDOM is not a major component of the OM in these particular caves and spring systems. The lack of humified DOM, indicated by the HIX values, implies that either terrestrial CDOM does not contribute to the overall carbon pool in these systems, or that the spectroscopic signature of terrestrial material is rapidly lost through unresolved abiotic and/or biotic processes. Although previous work has highlighted microbes being vital for DOM processing in cave and karst water systems (Farnleitner et al., 2005; Einsiedl et al., 2007; Simon et al., 2007; Birdwell and Engel, 2009), the spectroscopic properties identified for DOM in this study provide new opportunities for researchers to evaluate in situ microbial DOM signatures and sources in subsurface environments. Lastly, the work extends the application of simple absorbance and fluorescence spectroscopic metrics that can be used as indicators of CDOM properties. In particular, the results from these microbially dominated aquatic subsurface systems suggest that fluorescence indices can be used to describe a continuum of CDOM. Distinct CDOM groupings can be identified from BIX, FI and HIX values, as described in Table 2. The distinctions derived from absorbance spectral slopes are less clear for surface waters, but it is apparent that CDOM in cave and spring waters has different UV–Vis absorbance characteristics than CDOM from other, primarily photic, environments. These spectroscopic differences are likely due to a combination of environmental factors, including exposure to solar radiation, availability of organic carbon in the ecosystem of origin, in situ microbial activity, and mixing of autochthonous and allochthonous CDOM. However, further work needs to be done to differentiate among these various mechanisms. Acknowledgements The authors thank I. Warner, M. Lowry and R. Cook for equipment access and C. Schulz and K. Brannen for assistance with sample collection, processing and analysis. We thank two anonymous reviewers for helpful suggestions. The work was partially funded by the National Science Foundation (Chilean samples, EAR0544960) and Louisiana Board of Regents Support Fund Grants (contract NSF/LEQSF (2005)-Pfund-04) for the Italian cave and other hot spring samples. Associate Editor—E. A. Canuel References Aiken, G., McKnight, D.M., Harnish, R., Wershaw, R., 1996. Geochemistry of aquatic humic substances in the Lake Fryxell Basin, Antarctica . Biogeochemistry 34, 157–188. Alberts, J.J., Takács, M., 2004. Total luminescence spectra of IHSS standards and reference fulvic acids, humic acids and natural organic matter: comparison of aquatic and terrestrial source terms. Organic Geochemistry 35, 243–256. American Public Health Association, 1998. Standard Methods for the Examination of Water and Wastewater, 20th ed. American Public Health Association, Washington, DC. Anesio, A.M., Hollas, C., Granéli, W., Laybourn-Perry, J., 2004. Influence of humic substances on bacterial and viral dynamics in freshwaters. Applied and Environmental Microbiology 70, 4848–4854. Ariese, F., van Assema, S., Gooijer, C., Bruccoleri, A.G., Langford, C.H., 2004. Comparison of Laurentian fulvic acid luminescence with that of the hydroquinone/quinone model system: evidence from low temperature fluorescence studies and EPR spectroscopy. Aquatic Sciences 66, 86–94.

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