Carbon-13 nuclear magnetic resonance characterization of humic substances associated with salt marsh environments

The Science of the Total Environment, 101 (1991) 191-199 Elsevier Science Publishers B.V., Amsterdam

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CARBON-13 N U C L E A R M A G N E T I C R E S O N A N C E C H A R A C T E R I Z A T I O N OF HUMIC S U B S T A N C E S A S S O C I A T E D WITH S A L T M A R S H E N V I R O N M E N T S

Z. FILIP

Institut fi~r Wasser-, Boden- und Lufthygiene des Bundesgesundheitsamtes, Aussenstelle Langen, Paul-Ehrlich-Strasse 29, D-6070 Langen, Federal Republic of Germany R.H. NEWMAN

Chemistry Division, DSIR, Private Bag, Petone, New Zealand J.J. ALBERTS

The University of Georgia, Marine Institute, Sapelo Island, GA 31327, USA (Received December 8th, 1989; accepted January 29th, 1990)

ABSTRACT We have shown earlier that fresh and dead tissues of Spartina alterniflora, a plant species dominating Atlantic and Gulf coast salt marshes, contain humic substances which may be released into seawater. Carbon-13 NMR spectra have now been used to compare the chemical structures of these humic substances with humic matter extracted from mud in which S. alterniflora was growing. The results indicate that the mud humic matter differs from humic substances extracted from fresh plants primarily in the pattern of O-substitution of aromatic structures. However, humic substances associated with dead plants, or with fresh plants that had been incubated for a long time in seawater containing microorganisms indigenous to the mud, showed a great similarity to the humic matter from salt marsh mud.

INTRODUCTION

Salt marshes of the U.S. Atlantic and Gulf coasts are composed almost e n t i r e l y o f o n e v a s c u l a r p l a n t s p e c i e s : a s m o o t h c o r d g r a s s , S. alterniflora ( L o i s e l . ) . T h u s , S. alterniflora s h o u l d a l s o a c c o u n t f o r t h e p r i m a r y s o u r c e o f refractory carbon compounds, such as humic substances, in the mud and seawater of the salt marshes. Recent studies have listed the chemical composition and other properties of humic substances extracted directly from fresh S. alterniflora o r f r o m d e a d p l a n t d e b r i s ( A l b e r t s e t al., 1988; F i l i p e t al., 1988). Release of these humic substances into seawater by the action of microorganisms indigenous to salt marsh environments has also been described (Filip and A l b e r t s , 1988, 1989). The purpose of this study was to elucidate the stages of chemical transf o r m a t i o n b e t w e e n h u m i c s u b s t a n c e s a s s o c i a t e d w i t h e i t h e r f r e s h o r d e a d S.

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alterniflora tissues and the humic matter from salt marsh sediment (mud) using '3C-NMR spectroscopy. MATERIALSAND METHODS

Humic substances Stems and leaves of living green and standing dead S. alterniflora were collected from the marshes of Sapelo Island, Georgia. Mud was sampled at the site where the plants were growing. Details of procedures for isolating the humic substances are provided elsewhere (Filip et al., 1988; Filip and Alberts, 1988). In brief, humic substances were either extracted directly from dry samples of the plant material with a mixture of 0.1M NaOH and 0.1M Na4P2OT, acidified with HC1 to pH 1, dialyzed against deionized water and then freezedried or, alternatively, they were precipitated with HC1 from seawater in which the plant material was allowed to decompose for 10 months. The following humic substance preparations were used in the '3C-NMR spectroscopic studies: (a) from fresh plants; (b) from dead plants; (c) from mud; (d) from seawater containing fresh, sterile S. alterniflora; (e) from seawater containing fresh or dead S. alterniflora inoculated with epiphytic microflora; (f) from seawater containing fresh or dead S. alterniflora inoculated with the microbial population indigenous to the mud.

Nuclear magnetic resonance (NMR) Samples of ~ 0.1g of each humic substance were packed into cylindrical sapphire rotors (7 mm diameter) fitted with Kel-F end caps. The rotors were spun at 5 kHz in a magic-angle spinning (MAS) probe manufactured by Doty Scientific. Carbon-13 NMR sepectra were recorded at 50.3 MHz on a Varian XL-200 spectrometer. Each 5 gs proton preparation pulse was followed by a 2 ms cross-polarization contact time, 30ms of data acquisition and a 500ms pulse delay. Transients from between 4 × 104 and 4 x 105 contacts were averaged. Spin relaxation experiments were performed to confirm t h a t the pulse delay was adequate for recovery of the proton polarization, and that the contact time was adequate for cross-polarization to non-protonated carbon. All spectra in Figs 1-5 were plotted with vertical scales normalized on the height of the tallest peak. Signal areas were compared without correction for spinning sideband signals, because the MAS frequency was sufficient to suppress sideband signals to the level of noise. Assignments of 13C signals were assisted by use of a "dipolar dephasing" pulse sequence (Harbison et al., 1985), in which proton decoupling was interrupted for 42 its at the end of each contact. A 180° refocusing pulse was applied at the 13C-NMR frequency after one MAS rotation period, and data acquisition

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was delayed until completion of refocusing after an additional MAS rotation period. RESULTS AND DISCUSSION

Strong C-H dipole~iipole interactions lead to irreversible dephasing for signals associated with all methine and some methylene groups. Internal motion weakened the dephasing effects on methyl groups and some methylene groups. Signals from non-protonated carbon atoms were only slightly affected. A resultant spectrum is shown in Fig. 1. Signals suppressed by dipolar dephasing were recovered by subtracting the dipolar-dephased spectrum from a spectrum obtained by the normal crosspolarization pulse sequence. Vertical scales were adjusted to compensate for generalized loss of signal strength in dipolar dephasing, e.g. through imperfections in the 180° pulse. A resultant spectrum is shown in Fig. 2. 9

..~ -C-O-, -C-N-

i

100

5 / ppm

Fig. 1. Signals associated with relatively slow dipolar dephasing in the13C-NMR spectrum ofsolid humic substances released into seawater from dead S. alterniflora inoculated with the microbial population indigenous to the mud. I

HCOH

i

i

i

200

100

0

5/ppm

Fig. 2. Signals associated with relatively rapid dipolar dephasing in the 13C-NMR spectrum of the sample used to generate Fig. 1.

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Signal assignments shown in Figs 1 and 2 were based on published studies of other humic substances (Wilson, 1981, 1987; Newman et al., 1987, and others). The individual assignments are discussed here in categories based on the likely origins of the various chemical functional groups.

Proteins and peptides The NMR spectra shown in Figs3-5 contain a prominent signal at

a

c i

i

200

100

i

5 / ppm

0

Fig. 3. Carbon-13 NMR spectra of humic substances extracted from S. alterniflora. (a) Fresh plants, (b) dead plants, (c) mud.

6 I ppm

Fig. 4. C a r b o n - 1 3 N M R s p e c t r a o f h u m i c s u b s t a n c e s r e l e a s e d i n t o s e a w a t e r f r o m f r e s h p l a n t s o f S . alterniflora. (a) Under sterile conditions, (b) inoculated with the epiphytic microflora, (c) inoculated with the microbial population indigenous to the mud.

195

2oo

5 / ppm

Fig. 5. Carbon-13 NMR spectra of humic substances released into seawater from dead S. alterniflora inoculated with microbial populations indigenous to (a) plants, i.e. epiphytic microflora, (b) mud.

6 = 174 ppm, which is typical of the secondary amide linkages in proteins and peptides (Piotrowski et al., 1984). Further evidence for secondary amides is provided by a correlation between signal strength and the nitrogen content of the humic substances (Fig. 6). The N/C atomic ratios shown in Fig. 6 were calculated from the elemental analyses provided by Filip et al. (1988) and Filip and Alberts (1988). These authors also recognized the presence of amides in the S. alterniflora-related humic substances from Fourier transform infrared (FTIR) spectra. The correlation coefficient for a linear least-squares fit is 0.93. The intercept (0.037) is large enough to suggest that a fraction of the signal should perhaps be assigned to functional groups other than amides, and the fact that the slope (0.62) is less than unity supports this suggestion. Filip and Alberts (1989) also postulated that a significant portion of the nitrogen in some S. alterniflora humic substances might comprise amino acid nitrogen rather than amide-bound nitrogen. A similar correlation between signal strength and 015 O o

Ii

0"10

i 010 (Total N) / (Total [ )

0"15

Fig. 6. Correlation between the nitrogen/carbon ratio and signal strength at 6 = 174 for the humic sustances under test. The solid line represents the linear least-squares fit.

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nitrogen content has been reported for humic substances from municipal refuse disposed of in landfills (Newman et al., 1987). Proteins, peptides and their degradation products would also be expected to contribute towards the signal at about/i = 56, since the C-2 carbons of most amino acids show chemical shifts in this region (Stothers, 1972). The signal at = 56 in Figs 3-5 consists of a sharp component assigned to methoxyl groups (see below) with a broader underlying band. The strength of the broader band is similar to the strength of the signal at 5 = 174 in each of the eight spectra, as would be expected for secondary amide linkages. The C-2 carbon atoms of amino acids are directly bonded to hydrogen, and the NMR signals should therefore be suppressed by dipolar dephasing. This assignment is consistent with the absence of the broad band from Fig. 1.

Phenolics All spectra show signals across the range 5 = 103-160ppm, assigned to aromatic carbon. ~'Aromaticity", fa, is defined as carbon contained in aromatic rings as a fraction of total carbon; values were lowest for humic substances extracted directly from fresh plants (/ca = 0.26) or released from fresh plants in a culture inoculated with the epiphytic microbial population (fa = 0.27). All other values were clustered in a range fa = 0.39 + 0.02, with scatter of similar magnitude to experimental uncertainty. The sharp signal at /i = 56 is consistent with methoxyl substitution of aromatic rings, as in lignin. The assignment of this signal was tested in a dipolar dephasing experiment with a variable interruption interval (sample as in Figs I and 2). The decay of signal height at ~$ = 56 was separated into 60% Gaussian and 40% exponential contributions, with time constants of 16 and 190~s, respectively. The shorter time constant is typical of C-H bonds, e.g. in amino acids as in the assignment discussed above. The longer time constant appears too long for rigid C-H bonds, but too short for carbon lacking directly bonded hydrogen. The intermediate value of the time constant for dephasing resulted in this signal appearing in both Figs I and 2. Such behaviour is consistent with some freedom for internal motion of a methoxyl group around the O-C bond axis. Lignins isolated from grasses usually produce NMR signals in the region 148-162 ppm, assigned to O-substituted carbon atoms in aromatic rings (Nimz et al., 1981). The three structural units found in such lignins are: (a) guaiacyl units contributing signals from C-3 and C-4 at 5 = 148; (b) syringyl units contributing a signal from C-3 and C-5 at (f = 153; (c) 4-hydroxyphenyl units contributing a signal from C-4 somewhere in the vicinity of 5 = 162ppm. Figure 3a shows signals at all of these chemical shifts, with the signal at 5 = 148 dominating the region. Filip et al. (1988) also demonstrated the presence (in UV spectra) of lignin-linked aromatic structures in humic substances from fresh S. alterniflora. Guaiacyl structural units contain only one methoxyl group, but syringyl

197 units contain two. For a signal at 5 = 153 to be assigned to syringyl units, it should be associated with a contribution of similar strength at 5 = 56. Spectra in Figs 3b and 3c show a signal at 5 = 153 that is stronger than the signal at 5 = 148, but these spectra do not show a corresponding increase in the strength of the sharp component at 5 = 56. It is therefore unlikely that this signal is associated with lignin-like material.

Carbohydrates A band of signals in the vicinity of 5 = 75 can be assigned to -CH(OH)structures derived from carbohydrates, with perhaps some contribution from lignin sidechains. Spectra of solid hemicellulose show signals at 5 = 75 (Maciel et al., 1985), with weaker signals at 5 = 103 (anomeric carbon) and = 174 (carboxylic acid functional groups). The signal at 5 = 103 is weakest in those spectra showing the weakest signals at 5 = 75, i.e. Figs 3c and 4c, providing supporting evidence for the assignment. The signal at 5 = 174 would be obscured by the signal assigned primarily to amides. Fourier transform infrared spectra described by Filip and Alberts (1988) also indicate carbohydrates as constituents of S. alterniflora humic substances. Pakulski (1986) observed the release of different reducing sugars from S. alterniflora into a salt marsh environment.

Miscellaneous structures Some spectra showed weak signals at 5 = 195, assigned to ketones in conjugated structures. An alternative assignment to aldehydes can be eliminated, because the signal appears in Fig. 1 but not in Fig. 2. In the FTIR spectra of Filip and Alberts (1988), the ketone-related absorption appeared as a weak shoulder. Two spectra (Figs 3a and 4a) show weak signals at 5 = 183, assigned to carboxylic acids or quinones. A signal at (~ = 31 was assigned to methylene groups in long chains, e.g. lipid-like structures. The chains would have to be long enough for internal rotational motion to weaken the C-H dipole-dipole interaction, since the signal appears in Fig. 1 rather than Fig. 2.

General discussion Comparison of the spectra in Fig. 3 suggests that chemical transformations during the decay of standing plants must be more important than subsequent chemical transformations during incorporation of the dead plant materials into the mud. Transformations during the former period resulted in the following changes in NMR signals: (a) loss of a signal at (f = 183; (b) increase in strength of a signal at 5 = 153; (c) partial loss of resolved detail across the band from 5 = 103 to 5 = 129; and (d) partial loss of signal strength at 5 = 31

198 and 33. These observations are due to the decrease in the contents of aliphatic acids, carbohydrates, simple phenols, and some methylated aliphatic chains. A simultaneous increase of aromatic constituents in the humic substances may occur. Transformations during the latter period had little effect on the NMR signals (Fig. 3b and 3c). The humic substance released from fresh plant matter into seawater under sterile conditions (Fig. 4a) was similar to that extracted directly from fresh plant material (Fig. 3a), except for a higher ratio of amides to phenolic structures in the latter. Humic substances released into seawater from plant material inoculated with indigenous microbial populations were distinctly different; the NMR spectra were more similar to those for humic substance extracted from mud. These observations point to the importance of microbial activity in the transformation of S. alterniflora humic substances. Compared with humic substances released from fresh plant material, humic substances released from dead plant material showed a higher ratio of signal heights at 5 = 153 and 148, which is due to lignin-related aromatic structures. This is consistent with the extended period of degradative microbial activity in the decaying plant material. Whether or not the microbial population involved was indigenous to plants or mud seemed to have little effect on the chemical composition of the humic substances from S. alterniflora. CONCLUSIONS Carbon-13 NMR spectroscopic studies clearly indicate that: S. alterniflora makes a direct contribution to humic substances in salt marsh ecosystems; microbial activity during the decay of fresh plants results in considerable changes in chemical structures, particularly in enhancement of aromatic structural units; and S. alterniflora associated humic substances are incorporated in mud with little or no further transformation. Future studies will be carried out to elucidate the interactions of these humic substances with mineral constituents of mud, because these interactions may greatly influence the stability of refractory carbon in salt marsh environments. ACKNOWLEDGEMENTS This research was funded, in part, by the Deutsche Forschungsgemeinschaft, Bonn, and the Sapelo Island Research Foundation. The paper is a joint contribution of the i n s t i t u t fiir Wasser-, Boden- und Lufthygiene des Bundesgesundheitsamtes, the Chemistry Division, DSIR, and the UGA Marine Institute (No. 675). REFERENCES Alberts, J.J., Z. Filip, M.T. Price, D.J. Williamsand M.C. Williams, 1988. Elemental composition, stable carbon isotope ratios and spectrophotometricproperties of humic substances occurring in a salt marsh estuary. Org. Geochem., 12: 455-467.

199 Filip, Z. and J.J. Alberts, 1988. The release of humic substances from Spartina alterniflora (Loisel.) into sea water as influenced by salt marsh indigenous microorganisms. Sci. Total Environ., 73: 143-157. Filip, Z. and J.J. Alberts, 1989. Humic substances isolated from Spartina alterniflora (Loisel.) following long-term decomposition in sea water. Sci. Total Environ., 83: 273-285. Filip, Z., J.J. Alberts, M.V. Cheshire, B.A. Goodman and J.R. Bacon, 1988. Comparison of salt marsh humic acid with humic-like substances from the indigenous plant species Spartina alterniflora (Loisel.). Sci. Total Environ., 71: 157-172. Harbison, G.S., P.P.J. Mulder, H. Pardoen, J. Lugtenburg, J. Herzfeld and R.G. Griffin, 1985. High-resolution carbon-13 NMR of retinal derivatives in the solid state. J. Am. Chem. Soc., 107: 4809-4816. Maciel, G.E., J.F. Haw, D.H. Smith, B.C. Gabrielsen and G.H. Hatfield, 1985. Carbon-13 nuclear magnetic resonance of herbaceous plants and their components, using cross polarization and magic-angle spinning. J. Agric. Food Chem., 33: 185-191. Newman, R.H., B.K.G. Theng and Z. Filip, 1987. Carbon.13 nuclear magnetic resonance spectroscopic characterisation of humic substances from municipal refuse decomposing in a landfill. Sci. Total Environ., 65: 69~84. Nimz, H.H., D. Robert, O. Faix and M. Nemr, 1981. Carbon-13 NMR spectra of lignins. Holzforschung, 35: 16-26. Pakulski, J.D., 1986. The release of reducing sugars and dissolved organic carbon from Spartina alterniflora Loisel in a Georgia salt marsh. Estuarine Coastal Shelf Sci., 22: 384~395. Piotrowski, E.G., K.M. Valentine and P.E. Pfeffer, 1984. Solid-state, 13C, cross-polarization, "magicangle" spinning, NMR spectroscopy studies of sewage sludge. Soil Sci., 137: 194-203. Stothers, J.B., 1972. Carbon-13 NMR Spectroscopy. Academic Press, New York. Wilson, M.A., 1981, Applications of nuclear magnetic resonance spectroscopy to the study of the structure of soil organic matter. J. Soil Sci., 32: 167-186. Wilson, M.A., 1987. N.M.R. Techniques and Applications in Geochemistry and Soil Chemistry. Pergamon Press, Oxford.