Photochemical reactivity of dissolved lignin in river and ocean waters

© 1998, by the American Society of Limnology and Oceanography, Inc.

Photochemical reactivity of dissolved lignin in river and ocean waters

Stephen Opsahl and Ronald Benner Marine Science Institute, University of Texas at Austin, 750 Channelview Dr., Port Aransas, Texas 78373

Abstract The photochemical reactivity of dissolved lignin and photobleaching of dissolved organic matter (DOM) were

examined in riverine and open-ocean water samples. Approximately 75% of the total dissolved lignin in Mississippi River water was lost during 28 d of incubation in sunlight, mostly due to photooxidation. The remaining fraction of dissolved lignin was much less susceptible to photooxidation. About 90% of the dissolved lignin in river water was present as high-molecular-weight (HMW, > 1,000 Dalton) DOM. However, after exposure to sunlight, about 80% of the remaining lignin was present as low-molecular-weight (LMW, < 1,000 Dalton) DOM. An absolute increase in concentration of LMW lignin provided direct evidence for the phototransformation of macromolecular DOM to smaller molecules. The composition of riverine dissolved lignin also changed dramatically during photooxidation. The abundance of syringyl relative to vanillyl phenols decreased twofold, while concentrations of vanillic acid relative to vanillin, (Ad/Al)v, increased fourfold. The increase in (Ad/Al)v was found exclusively in the photooxidized LMW DOM. The photochemically altered composition of riverine dissolved lignin was consistent with the composition of oceanic dissolved lignin, The dissolved lignin in HMW DOM from the equatorial Pacific Ocean was highly resistant to photooxidation. These findings suggest that photochemical reactions play a prominent role in determining the composition and reactivity of terrigenous DOM in the ocean,

Approximately 0.25 X 1015 g of terrigenous dissolved organic matter (DOM) is discharged to the oceans annually (Meybeck 1982), yet ambient concentrations throughout the oceans are low (Opsahl and Benner 1997), indicating a short residence time and rapid cycling in the ocean, Large gradients of terrigenous DOM observed along coastal zones (Moran et al. 1991) suggest that much of the remineraliza- tion of terrigenous DOM takes place in ocean margins. Both photochemical and microbial oxidation are important removal mechanisms for riverine DOM (Kieber et al. 1990; Benner et al. 1995; Miller and Zepp 1995; Amon and Benner 1996b), and each has the potential to selectively alter its inherent biochemical signature. The relative importance of these processes varies depending on the biochemical composition of the DOM and environmental conditions. For example, microbial degradation is the dominant removal mechanism for DOM in sediment-laden rivers, whereas rates of photochemical oxidation could be quite high in river plumes and coastal waters, where terrigenous DOM is exposed to high levels of solar radiation. To better characterize and un- derstand the reactivity of terrigenous DOM in the marine environment, photochemical and microbial transformations of this DOM need to be identified.

Terrigenous DOM yields a variety of oxidation products when subjected to natural levels of sunlight. Direct conver- sion of DOM to CO, and CO represents a major sink for terrigenous DOM (Kieber et al. 1990; Valentine and Zepp 1993; Miller and Zepp 1995). DOM photoproducts include

Acknowledgments We are grateful to J. Hedges, M. Goñi, and an anonymous re-

viewer for their helpful comments on this manuscript. We thank Dave Kieber for the use of quartz flasks and the incubation rack on the equatorial Pacific cruise. We also thank Brenda Black for assis- tance with sample processing and laboratory analyses. This research was sponsored by National Science Foundation grants OCE 943843 and DEB 9420652. This is contribution 1047 from the University of Texas at Austin Marine Science Institute in Port Aransas, Texas.

various low-molecular-weight organic compounds that are rapidly utilized by microorganisms (Mopper and Stahovec 1986; Kieber et al. 1989; Mopper et al. 1991; Wetzel et al. 1995). In addition to direct transformations, specific components of DOM that absorb ultraviolet (UV) light may act indirectly as a photosensitizer that catalyzes photochemical reactions with other constituents (Zafiriou et al. 1984; Zepp 1988). Although many photoreactions involving different DOM constituents have been studied, the extent of degradation and compositional alterations of terrigenous DOM are not well understood.

Lignin is a phenolic polymer unique to vascular plants (Sarkanen and Ludwig 1971) and an important component of riverine DOM (Ertel et al. 1984, 1986). The unique biochemical signature of lignin provides a means to “finger- print” riverine DOM (Ertel et al. 1984, 1986) and to quantify its concentration in dilute matrices including ocean water (Meyers-Schulte and Hedges 1986; Opsahl and Benner 1997). In addition to establishing a terrigenous origin, lignin oxidation products also provide information regarding the botanical source and diagenetic state of the associated organic matter (Hedges and Mann 1979; Ertel and Hedges 1984; Ertel et al. 1984). Laboratory studies have shown that lignin is susceptible to photooxidation (Castellan et al. 1987; Vanucci et al. 1988), and the lignin component of dissolved humic substances is known to be photoreactive (Ertel 1990). However, little is known regarding selective compositional alterations that may result from the photochemical oxidation of dissolved lignin and the consequences such changes have on its molecular signature.

In this study, an experimental approach was taken to ex- amine the potential for photochemical alterations of DOM and its associated dissolved lignin component in riverine and open-ocean water samples. We consider whether changes in the dissolved lignin component of riverine DOM due to photochemical oxidation can help explain the observed composition and reactivity of dissolved lignin in the ocean.

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Materials and methods

Exposure of Mississippi River water to sunlight-Water was collected from the Mississippi River about 50 km up- stream from its discharge into the Gulf of Mexico. The water was filtered through a 0.2-µm filter to remove particulates. The filtration was about 50% efficient in removing bacterial cells, leaving approximately 0.5 X l06 cells ml-1. In our experience, these filters typically remove a larger percentage (-95%) of the bacterial population, but these samples clearly contained a relatively large bacterial community. For samples exposed to light, large (1.0 liter) quartz tubes were filled with water and sealed with teflon-lined stoppers. This treatment was used to approximate in situ conditions in which both microbial and photochemical processes occur. For dark samples, Pyrex tubes covered in aluminum foil were used. This treatment excludes light so that only microorganisms are active. All tubes were placed on racks in an outdoor water tank with flowing seawater for temperature control. Tubes were incubated at the surface for maximum light exposure. Duplicate samples were harvested at 0, 5, 14, 21, and 28 d. Subsamples were collected for bacterial abundance and UV absorbance. The remaining sample was evaporated to dryness using a rotoevaporator, and the dried material was collected for chemical analysis. UV-A and UV-B irradiance were measured with an IL1700 radiometer (International Light) equipped with SUL 033 and SUL 240 broadband de- tectors for measuring UV-A (315-390 nm) and UV-B (275- 3 10 nm), respectively.

Separation of river water into high- and low-molecular- weight fractions (HMW, LMW) was accomplished using ultrafiltration as described by Benner (199 1) and Benner et al. (1997). For the untreated river water sample, 50 liters was processed, first through a 0.2-µm filter to remove particles and then through polysulfone membranes with a 1,000-Dal- ton pore size. For the photooxidized river water sample, 2 liters of light-exposed water (as described above) was collected at the end of the experiment (28 d) for ultrafiltration. The volume of water was brought up to 20 liters using Milli- Q Plus UV water, and this volume was then ultrafiltered in a manner similar to that used in the initial river water sample. An organic carbon mass balance showed 97 and 99% carbon recovery in the combined fractions (HMW + LMW) for the initial and photooxidized river water samples, respectively.

Exposure of Pacific Ocean water to sunlight- A large (1,000 liter) water sample was collected from the equatorial Pacific Ocean (2.00°S, 140.00°W) from the oxygen mini- mum layer at a 400-m depth. The water was filtered through a 0. l-µm pore size filter, and the HMW (> 1,000 Dalton) was concentrated to 1.0 liter using tangential-flow ultrafiltration (Benner et al. 1997). Half the water sample was placed in quartz tubes, and the other half was placed in aluminum foil-wrapped polycarbonate bottles. Water samples were placed in a deckboard incubator at in situ water temperatures (ca. 27°C) and exposed to 4 d of sunlight. Subsam- ples were collected for UV-absorbance measurements, and the remaining concentrate was dried for subsequent chemical analysis. Total light flux was measured using an IL1700 ra-

OH

Fig. 1. Names, structures, and abbreviations of vanillyl, syringyl, and cinnamyl phenols derived from the CuO oxidation of lignin.

diometer (International Light) equipped with a broadband silicon detector (SUD 038), quartz diffuser, and neutral den- sity filter.

Dried samples were prepared for lignin analysis using the CuO oxidation and extraction scheme of Hedges and Ertel (1982) with the modifications of Goni and Hedges (1992) and Opsahl and Benner (1995, 1997). Lignin-derived phenols were separated using a Hewlett Packard 5890A gas chromatograph fitted with an HP-5MS capillary column (30 m, 0.25-mm inner diameter). A Hewlett Packard 5972 mass selective detector was used to positively identify individual phenols based on comparison with standards. Quantification was achieved using selected ion monitoring and ethylvanillin as an internal recovery standard. Chemical structures of the major lignin-derived CuO oxidation reaction products reported in this study are given in Fig. 1. The vanillyl and syringyl phenols are among the most abundant lignin-derived CuO oxidation products, and the sum of these six phenols were used to represent lignin in this study (Opsahl and Benner 1995).

UV absorbance was measured using a Shimadzu UV16OU spectrophotometer and l- or 5-cm quartz cells. Milli-Q Plus UV water was used to zero the instrument. Elemental analyses (C, H, and N) were made using a Carlo Erba EA1108 elemental analyzer. Bacterial abundance was measured ac- cording to the method of Porter and Feig (1980).

Photooxidation of lignin 1299

Table 1. Concentrations (µg liter-l) and exponential decay constants (k; d-l) of individual lignin phenols from bottle incubations of Mississippi River water.*

Days VAL VON VAD SAL SON SAD CAD FAD

Dark 0 5

14 21 28

Light 0 5

14 21 28

k X 10-2 (d-l)

1.97 1.44 2.46 1.81 1.52 2.32 1.58 1.28 1.39 1.51 1.38 2.36 1.69 1.63 2.47

1.97 1.44 2.46 1.86 1.65 1.18 0.47 0.42 1.06 0.93 1.89 0.91 1.00 0.73 0.35 0.28 0.57 0.53 1.15 0.50 0.60 0.28 0.27 0.15 0.31 0.35 0.83 0.30 0.42 0.18 0.24 0.11 0.24 0.42 1.03 0.20 0.30 0.16 0.26 0.10 4.27 6.07 3.50 3.74 4.25 5.43 2.11 5.74

1.86 1.61 1.55 1.44 1.46

1.65 1.18 0.47 0.42 1.66 1.11 0.45 0.47 1.60 1.06 0.40 0.35 1.48 1.15 0.45 0.40 1.50 1.01 0.47 0.45

* Abbreviations provided in Fig. 1. A simple model of exponential decay (c1 = c0 X e-kt) was used to estimate the decay constant k, where c, is the phenol concentration at a given time t, and c0 is the initial phenol concentration.

Results and discussion

Photooxidation of dissolved lignin and DOM photobleaching- concentrations of individual vanillyl and syringyl phenols ranged from about 1.2 to 2.5 µg liter-1 (Table l), whereas cinnamyl phenols were found in much lower concentrations (0.4-0.5 µg liter-1). This distribution of individual phenols is similar to that reported from humic substances isolated from the Amazon River (Ertel et al. 1986) and ultrafiltered DOM isolated from both the Amazon and Mississippi Rivers (Opsahl and Benner 1997). The relatively high proportion of syringyl phenols and the presence of sub- stantial cinnamyl phenols reflect a major contribution of nonwoody angiosperm plant tissues to Mississippi River DOM.

The dissolved lignin component of Mississippi River water decreased in concentration by about 80% after 28 d of sunlight exposure (Fig. 2). In contrast, concentrations of dis-

Fig. 2. Lignin phenol concentrations (measured as the sum of the six vanillyl and syringyl phenols) in light (open symbols; y = 8.81x e(-0.0582x), R = 0.97, P < 0.05) and dark (solid symbols; y = 9.84 + -0.058x, R² = 0.41, P > 0.05) treatments. Each value represents the average of duplicate incubations.

solved lignin incubated in the dark decreased only 16% during the same time interval. The observed decreases in lignin concentration in the dark were attributed to microbial degradation, although it is conceivable that some dissolved lignin adsorbed to the container walls. The large difference between light and dark incubations clearly demonstrated that sunlight played a major role in the degradation of dissolved lignin. Under conditions that were optimized for photochemical transformations, lignin loss in the light treatment ex- ceeded that of the dark controls by 4.5-fold. The decrease in lignin concentration due to photooxidation was described by a simple exponential decay model (R = 0.97, P < 0.01; Fig. 2), with little change occurring the last 7 d of incubation. These results suggest that the fraction of dissolved lignin remaining at the end of the incubation was more resistant to photochemical and microbial alteration than the majority of lignin, which had degraded relatively rapidly.

Exposure of Mississippi River DOM to natural levels of sunlight for 28 d resulted in significant photobleaching of the DOM (Fig. 3). The percent absorbance lost in the UV portion of the spectrum was generally high, ranging from 25 to 40% after 5 d and from 40 to 60% after 28 d. Corre- sponding dark controls showed no change in absorbance during the incubation. Highest levels of photobleaching occurred between 320 and 360 nm. Photobleaching of DOM based on loss of UV absorbance has been reported for a wide variety of riverine waters (Kieber et al. 1990; Valentine and Zepp 1993; Miller and Zepp 1995). In those three studies, the production of CO, and CO from DOM was strongly correlated with absorbance bleaching. In this study, the large loss of lignin from the DOM pool was not correlated (P > 0.05) with the extensive absorbance bleaching. Although losses in dissolved lignin and absorbance are related to compositional changes in the DOM pool, it appears that absorbance measurements alone can not be used to gauge the extent of lignin oxidation.

The dissolved lignin component of waters from the equatorial Pacific provided an interesting comparison with that from the Mississippi River. Whereas dissolved lignin con-

Opsahl and Benner

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40

20

0

Fig. 3. Percentage of UV absorbance lost in filtered water from the Mississippi River after exposure to sunlight for 5 d (open squares) and 28 d (open circles). Dark controls after 28 d of incubation are represented by solid circles. Each value represents the average of duplicate incubations.

centrations in the Mississippi River dropped 37% during the first 5 d of incubation, lignin concentrations in the Pacific water sample did not change substantially after 4 d of exposure to sunlight (Table 2). This demonstrated the presence of dissolved lignin in the HMW fraction of seawater DOM that is resistant to photochemical oxidation. Such negligible levels of photochemical oxidation were also observed for riverine DOM that had undergone extensive photochemical oxidation (Fig. 2). Similarly, the UV-absorbing component of this equatorial Pacific water sample was quite resistant to photobleaching (Fig. 4), but the absorbance of Mississippi River water exposed to sunlight was reduced about 25-40% after 5 d of light exposure (Fig. 3). The absorbance data were consistent with the lignin data, both of which indicate little photochemical reactivity of HMW DOM in seawater from the Pacific Ocean.

Photochemical alterations of dissolved lignin: Riverine DOM- The ratio of the syringyl to vanillyl phenols (S/V) provides useful information about the taxonomic source of vascular plant-derived DOM because syringyl phenols are relatively abundant in angiosperm tissues and absent from gymnosperm tissues (Hedges and Mann 1979). In this study, Mississippi River dissolved lignin had an initial S/V ratio of about 0.8 (Fig. 5), reflecting an abundance of angiosperm- derived DOM in this temperate drainage basin. This S/V

Table 2. Concentration and composition of dissolved lignin in equatorial Pacific Ocean ultrafiltered DOM (>1,000 Dalton) after 4-d incubation at sea-surface temperatures. Average integrated daily light flux was 271 W cm-2 d-l.

Fig. 4. UV absorbance (350 nm) during a 4-d incubation of HMW (> 1,000 Dalton) dissolved organic matter (DOM) isolated from equatorial Pacific Ocean water using tangential-flow ultrafiltration. Each value represents a single measurement from a subsam- ple taken during the time course (y = 0.16 + 0.0024x, R² = 0.49, P > 0.05).

ratio is within the range of previously reported values for Mississippi and Amazon River ultrafiltered DOM (Opsahl and Benner 1997), but it is higher than that reported for Amazon River humic fractions, which were 0.45 and 0.27 for humic and fulvic acids, respectively (Ertel et al. 1986). This ratio is also higher than that reported for open-ocean ultrafiltered DOM (~0.25; Opsahl and Benner 1997). During exposure to sunlight, S/V ratios of Mississippi River water decreased significantly (P < 0.01), whereas no significant change was observed in the dark treatment (P > 0.05). This decrease demonstrated that the syringyl subunits as a group were more susceptible to photochemical alteration than vanillyl subunits.

The S/V ratios of decomposing vascular plant tissues in particulate material have been examined to determine the extent to which biodegradation may alter this source indicator (Hedges et al. 1988; Benner et al. 199 1; Opsahl and

0.6

Fig. 5. Ratio of syringyl to vanillyl phenols, S/V, in light (open symbols; y = 0.78 - 0.012x, R² = 0.90, P < 0.05) and dark (solid symbols; y = 0.86 - 0.0029x, R² = 0.06, P > 0.05) treatments. Each value represents the average of duplicate incubations.


Days

Fig. 6. Ratio of vanillic acid to vanillin, (Ad/Al)v, in light (open symbols; y = 1.24x e (0.0415x), R = 0.97, P < 0.05) and dark (solid symbols; y = 1.11 + 0.0114x, R² = 0.23) treatments. Each value represents the average of duplicate incubations.

Benner 1995). Although small changes in S/V ratios have been reported, only degradation of woody tissues by soft-rot fungi has shown decreases in S/V ratios that are comparable in magnitude to the changes reported here for photochemical alteration (Hedges et al. 1988). However, these fungi are most prevalent in terrestrial environments and do not con- tribute to the degradation of dissolved lignin in seawater. Decomposition studies examining the susceptibility of dissolved lignin to selective microbial degradation have not yet been undertaken. In this study, incubation of Mississippi River water in the dark bottles for 28 d revealed no significant changes in S/V due to biological degradation (Fig. 5), but this cannot preclude selective changes that may occur over longer time scales. Although the potential of marine microorganisms to selectively alter S/V ratios of dissolved lignin remains unclear, it is apparent that photochemical oxidation can induce large changes that can occur relatively rapidly.

The ratio of syringaldehyde to syringic acid, (Ad/Al)s, also increases during microbial oxidation (Hedges et al. 1988; Opsahl and Benner 1995). In this study, initial (Ad/ Al)s ratios of 0.6 were variable in both light and dark treatments, and no clear trend was apparent (Fig. 7). The reason for this difference in behavior of syringyl and vanillyl phenols is not known. Vanillyl and syringyl phenols are struc- turally similar and differ only by the presence of an additional methoxyl group on syringyl phenols (Fig. 1). Unlike (Ad/Al)v, it appears that (Ad/Al)s will not have the added utility of reflecting photochemical alterations of dissolved lignin. Notably, these results were quite similar to those reported for lignin degradation by soft-rot fungi (Hedges et al. 1988), in which a large increase in (Ad/Al)v accompanied degradation, but minimal changes were observed in (Ad/ Al)s. As with S/V, biochemical changes in Ad/Al ratios due to photochemical oxidation appeared to closely parallel those reported for soft-rot fungal decay of lignin. The strong parallel in patterns of decay may not be fortuitous since reactive oxygen is involved in both degradation pathways.

Ratios of vanillic acid to vanillin, (Ad/Al)v, for fresh plant Two cinnamyl phenols, p-coumaric acid and ferulic acid, tissues (0.1-0.5) are known to increase during microbial are additional CuO oxidation products of lignin that are par- degradation (Hedges et al. 1988; Benner et al. 1991; Opsahl ticularly abundant in grasses and many herbaceous tissues. and Benner 1995), providing a useful diagenetic indicator of Concentrations of both compounds changed only slightly in biological oxidation (Ertel and Hedges 1984; Ertel et al. the dark incubations (Table l), whereas p-coumaric acid con- 1984). However, the potential for sunlight as an additional centrations decreased by ~45% and ferulic acid by about mechanism responsible for changes in (Ad/Al)v ratios has 75% in the tubes exposed to sunlight (Table 1). This dem- not been previously examined. The initial (Ad/Al)v of Mis- onstrates that ferulic acid is much more susceptible to pho- sissippi River water was about 1.2 (Fig. 6), within the range tochemical oxidation than p-coumaric acid. These two phe- reported for dissolved humic substances of Amazon River nols differ by the presence of a methoxyl group (Fig, l), and (Ertel et al. 1986) and was indicative of highly oxidized this may explain their different susceptibility to photochem- organic material. During the experiment, (Ad/Al)v ratios in- ical oxidation. Corresponding increases in the ratio of cin- creased greatly in the light relative to the dark, indicating namyl to vanillyl phenols (C/V) after 3 weeks were evident, that photochemical oxidation was responsible for the ob- due to photochemical oxidation (Fig. 8), indicating that, as served changes. An (Ad/Al)v ratio of 4.0 is at the upper end a group, cinnamyl phenols were slightly more resistant to of values reported for riverine, coastal, and open-ocean photochemical oxidation than vanillyl phenols. This provides DOM (Ertel et al. 1986; Moran et al. 1991; Opsahl and Ben- an interesting contrast to previously reported decreases in C/ ner 1997). Although microbial and photochemical reactions V, which occurred during the biological degradation of vas- are fundamentally different processes, both can be respon- cular plant tissues. Whether or not microbial degradation is sible for increasing this diagenetic indicator of oxidative responsible for selectively changing the C/V of dissolved degradation. lignin, photochemical alteration should be considered a like-

Fig. 7. Ratio of syringic acid to syringaldehyde, (Ad/Al)s, in light (open symbols) and dark (solid symbols) treatments. Each value represents the average of duplicate incubations.


Days

Fig. 8. Ratio of cinnamyl to vanillyl phenols, C/V, in light (open symbols) and dark (solid symbols) treatments. Each value represents the average of duplicate incubations.

ly mechanism for elevating C/V ratios in naturally occurring samples.

The susceptibility of lignin phenols to photochemical oxidation was examined by calculating exponential decay constants from the light incubations after subtraction of dark controls (Table 1). Within the vanillyl and syringyl phenol families, no discernible trend was evident among the acid, aldehyde, and ketone subcomponents. For example, vanillic acid was the least chemically reactive vanillyl phenol, whereas syringic acid was the most reactive syringyl phenol. An overall stability series from most to least photochemi- tally reactive was VON > FAD > SAD > VAL > SON > SAL > VAD > CAD (see Fig. 1 for acronym definitions).

Photochemical alterations of dissolved lignin: Open- ocean DOM- Dissolved lignin from the equatorial Pacific was not susceptible to photochemical oxidation, and no photochemically induced changes in S/V or (Ad/Al)v were observed (Table 2). In contrast, large differences in lignin concentration, S/V, and (Ad/Al)v were measured in riverine DOM after a comparable incubation period (Table 1; Figs. 1, 5, 6). The initially high (Ad/Al)v and low S/V relative to fresh riverine DOM indicate that the dissolved lignin component of open-ocean DOM had undergone prior oxidative degradation. Elevated (Ad/Al)v and reduced S/V were consistent with the photochemically driven changes seen in Mis- sissippi River DOM. These trends suggest that photooxidation plays an important role in the cycling of terrigenous DOM in the ocean.

Lignin photoreactions- Although specific mechanisms for the photooxidation of dissolved lignin cannot be deter- mined in this study, certain indirect evidence for key reactions may be useful in explaining the observed compositional changes. For example, shifts in the S/V ratios imply that aryl methoxyl groups (Fig. 1) may be photochemically la- bile. The susceptibility of methoxyl groups to photochemical oxidation is also supported by the rapid photooxidation of ferulic acid relative to p-coumaric acid. These two phenols

differ only by the presence of an additional methoxyl group bound to ferulic acid (Fig. 1). The large increase in (Ad/Al)v indicates that the side chains of macromolecular lignin are also susceptible to photochemical alteration. The high levels of photobleaching indicate loss of aromaticity and are sug- gestive of ring cleavage as an additional reaction in the photooxidation of dissolved lignin. Oxygen mediated photochemical degradation of methoxyl groups, side chains, and aromatic rings has been reported in previous laboratory studies (Fengel and Wegener 1984). Extreme photobleaching and complete removal of lignin was observed in seagrass detritus exposed to high levels of sunlight (Opsahl and Benner 1993). Whether most lignin is directly remineralized or first converted to LMW organic compounds remains unknown. Regardless, both degradative pathways have important implications for the reactivity of riverine DOM and can modify the associated lignin phenol signature.

Rates of dissolved lignin photooxidation in Mississippi River water were reduced dramatically toward the end of the incubation (Fig. 2), indicating that a fraction of dissolved lignin was resistant to photooxidation. Similarly, dissolved lignin in equatorial Pacific DOM was highly resistant to photochemical oxidation. It is possible that within the lignin macromolecule there are photochemically resistant inter- monomeric bonds that allow a fraction of the original bio- macromolecule to persist. However, physical shielding by other DOM components or the formation of photochemically resistant bonds between lignin and other compounds are also explanations that can account for the observed results. Low- er rates of lignin oxidation may also result from loss of photochemical sensitizers. Photosensitizers are light-absorbing compounds that participate in photochemical transformations of other compounds (Zepp 1988). It is possible that photosensitizers are relatively abundant in riverine water but are biologically and/or photochemically degraded over time. This would explain the declining rate of lignin oxidation observed during the photooxidation of Mississippi River water. Photosensitizers may be much less abundant in the open- ocean, which may explain the lack of lignin photooxidation in this environment.

Photochemical oxidation and the size distribution of DOM- Mass balance calculations indicate that 103% of the total dissolved lignin in Mississippi River water was ac- counted for in the HMW concentrate and the LMW per- meate. About 90% of the dissolved lignin in the initial river water was found in the HMW fraction (Table 3), demonstrating the predominance of macromolecular lignin in riverine DOM. Extensive photochemical oxidation was accompanied by a major shift in size distribution, in which 79% of the remaining dissolved lignin was found in the LMW fraction. The absolute increase in concentration of LMW lignin demonstrated that LMW lignin was formed from HMW lignin. The absolute increase in LMW lignin also indicated that LMW lignin was more resistant to photooxidation than HMW lignin. The existence of a photochemically refractory LMW fraction is further supported by the observation that rates of photooxidation decreased during the incubation period (Fig. 2). Thus, the lignin macromolecule characteristic


Table 3. The effect of sunlight on lignin concentration and composition in high-molecular-weight (HMW, > 1,000 Dalton) and low- molecular-weight (LMW, < 1,000 Dalton) fractions of Mississippi River water. Average midday irradiance (combined UV-A and UV- B) was 27.9 W m-2.

Lignin phenol concentration (µg liter-1)*

Initial water

HMW LMW

9.76 1.09

After 28 d of sunlight exposure

HMW LMW

0.69 2.55

(Ad/Al)v 1.42 1.35 1.07 11.69 (Ad/Al)s 0.99 0.39 0.38 1.49 S/V 0.86 0.44 0.13 0.09

* Calculated as the sum of the six vanillyl and syringyl phenols; (Ad/Al)v, ratio of vanillic acid to vanillin; (Ad/Al)s, ratio of syringic acid to syringaldehyde; S/V, ratio of syringyl to vanillyl phenols.

of riverine DOM underwent photochemically driven reductions in both size and reactivity.

Changes in lignin composition of the HMW and LMW size fractions revealed additional information regarding the photochemical alteration of dissolved lignin and its accom- panying diagenetic signature. The (Ad/Al)v ratios in the HMW and LMW fractions of the initial river water were not substantially different, indicating similar levels of oxidation (Table 3). However, sunlight exposure caused a dramatic increase in the (Ad/Al)v of the LMW fraction, demonstrating that the remaining lignin in the LMW fraction was highly oxidized. Photochemical oxidation had a similar effect on (Ad/Al)s, resulting in a fourfold increase in the (Ad/Al)s of the LMW fraction. The (Ad/Al)v and (Ad/Al)s in the HMW fraction decreased compared to the original water sample. It is possible that more highly oxidized molecules on the pe- riphery of the HMW dissolved lignin complex were pref- erentially removed, leaving a small, less oxidized fraction. Overall, most of the remaining dissolved lignin consisted of highly oxidized small molecules, which appear to be the major product of extensive photochemical oxidation.

The S/V of the HMW fraction in the initial water was about twofold higher than that of the LMW fraction (Table 3). This difference may be indicative of more diagenetically altered material in the LMW fraction, although this was not apparent from (Ad/Al) ratios. Photochemical oxidation decreased S/V ratios in both the HMW and LMW fractions. A lower S/V in the LMW fraction would be expected because the (Ad/Al)v ratios indicated it was more diagenetically altered than the HMW fraction. A decrease in S/V in the HMW fraction demonstrated selective photochemical degradation of syringyl phenols relative to vanillyl phenols. Al- though the HMW material did not increase in oxidative state, changes in S/V clearly indicated that diagenetic alterations were occurring in the HMW fraction because of photochemical oxidation.

Tracing riverine DOM- The transport of riverine DOM to the ocean accounts for the largest flux of reduced carbon from the terrestrial to the marine environment and thus represents an important pathway in the global carbon cycle

(Hedges et al. 1997). To accurately use lignin as a biomarker to trace riverine organic matter, the various mechanisms responsible for its removal need to be examined for their potential to selectively degrade lignin and alter its relative abundance. For example, losses due to photochemical oxidation may occur relatively rapidly compared to biological decomposition in river plumes, where buoyant water masses lose their sediment load and remain exposed to sunlight. The potential for rapid photooxidation of dissolved lignin is supported by large onshore-offshore concentration gradients measured along continental shelves (Moran et al. 1991) and the low concentrations found in the open ocean (Meyers- Schulte and Hedges 1986; Opsahl and Benner 1997). In contrast, studies on the biodegradation of plant tissues in aquatic environments have shown that lignin is either relatively resistant to decomposition or degrades at a rate similar to the associated organic matter (Opsahl and Benner 1995). Further studies should attempt to resolve the relative importance of photochemical and biological degradation of terrigenous DOM in the ocean. These processes ultimately determine the relative abundance of lignin in terrigenous DOM, and a greater understanding will refine estimates of terrestrial con- tributions to DOM in the ocean.

To date, chemical characterizations of DOM have not provided specific parameters that reflect photochemical oxidation. However, S/V and (Ad/Al)v ratios derived from lignin phenol analyses hold promise as new indicators of photochemical transformations. Although complete recovery of dissolved lignin from ocean waters has not yet been achieved, Opsahl and Benner (1997) reported lignin phenol compositions for the HMW fraction (> 1,000 Dalton) of DOM from the Atlantic and Pacific Oceans. The S/V of open-ocean HMW DOM is lower than that reported for major river systems (Opsahl and Benner 1997). The photochemically induced reductions in S/V observed in this study represent a mechanism that can account for this transformation within the ocean. Open-ocean (Ad/Al)v ratios for the HMW fraction were quite similar to that reported for riverine DOM and were similar to the HMW fraction remaining after photooxidation (Table 3). It was the LMW fraction that increased in (Ad/Al)v as a result of photooxidation, but this size fraction of DOM can not be isolated from seawater using ultrafiltration. Based on results from this study, we spec- ulate that the LMW fraction in open-ocean DOM may have a higher (Ad/Al)v than the HMW fraction. If changes in S/ V and (Ad/Al)v prove to result primarily from photochemical processes, then these lignin-derived parameters will be powerful new indicators of photochemically transformed DOM.

DOM transformations- The ability to detect and quantify lignin in different molecular size fractions of DOM provided specific evidence that a dissolved macromolecule can un- dergo a decrease in molecular size during photochemical oxidation yet retain its molecular identity. The transformation in size was also followed by a decrease in reactivity. This is consistent with the size reactivity continuum model of marine DOM proposed by Amon and Benner (1996a), in which a general reduction in the size of the marine DOM pool is accompanied by a reduction in reactivity. If such


features are found to hold true for additional DOM components, then these results carry broad implications for the impact of photochemical and biological transformations of DOM on the carbon cycle of the ocean.

References

Received: 3 September 1997 Accepted: 24 February 1998

Amended: 9 April 1998

Photochemical reactivity of dissolved lignin in river and ocean waters

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