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Humic substances isolated from Spartina alterniflora (Loisel.) following long-term decomposition in sea water
The Science of the Total Environment, 83 (1989) 273-285 Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
273
HUMIC SUBSTANCES ISOLATED FROM SPARTINA ALTERNIFLORA (LOISEL.) FOLLOWING LONG-TERM DECOMPOSITION IN SEA WATER
Z. FILIP Institut fiir Wasser-, Boden- und Lufthygiene des Bundesgesundheitsamtes, Aussenstelle Langen, Paul.Ehrlich-Strasse 29, D-6070 Langen (Federal Republic of Germany) J.J. ALBERTS The University of Georgia, Marine Institute, Sapelo Island, GA 31327 (U.S.A.) (Received December 1st, 1968; accepted January 12th, 1989)
ABSTRACT Fragments of fresh or dead Spartina alterniflora which were decomposed for 10 months in sea water yielded appreciable amounts of humic substances when extracted with alkali under nitrogen. The plant materials inoculated with mixed populations of epiphytic or mud microorganisms indigenous to the salt marsh yielded up to 168% more humic substances than controls. The results of elemental analyses, including atomic ratios for some elements, and also UV and Fouriertransform infrared (FrIR) spectroscopy investigations, indicate a great similarity between humic substances from the decomposing S. alterniflora and salt marsh humic acid. Calculations based on the experimental data indicate a potential annual input of ~ 330 kg of fresh Spartina-related humic substances per hectare of salt marsh. INTRODUCTION On s e d i m e n t i n g s h o r e l i n e s in t e m p e r a t e l a t i t u d e s , l a r g e a r e a s of t h e u p p e r i n t e r t i d a l zone a r e o f t e n d o m i n a t e d by S p a r t i n a m a r s h e s (Mann, 1988). Also, t h e p r i m a r y p r o d u c t i o n o f b i o m a s s o c c u r r i n g in t h e s a l t m a r s h e s of t h e A t l a n t i c a n d G u l f c o a s t s of t h e U.S.A. t a k e s p l a c e m a i n l y in t h e v a s t s t a n d s of t h e s m o o t h c o r d g r a s s S p a r t i n a alterniflora (Loisel.). T h e losses of dissolved o r g a n i c m a t t e r f r o m living s h o o t s of S. alterniflora d u r i n g t i d a l s u b m e r g e n c e in t h e s a l t m a r s h e s , a n d f r o m detritus, i.e. d e a d p l a n t f r a g m e n t s a n d a s s o c i a t e d m i c r o o r ganisms which are almost permanently submerged, represent an important i n p u t of n u t r i e n t s i n t o e s t u a r i n e food webs (Teal, 1962; H a i n e s a n d H a n s o n , 1979; P o m e r o y et al., 1981; P a k u l s k i , 1986). S i m u l t a n e o u s l y , n e w f o r m s of r e f r a c t o r y o r g a n i c m a t t e r , s u c h as h u m i c s u b s t a n c e s , c a n be f o r m e d by a b i o t i c p o l y m e r i z a t i o n o f simple a l i p h a t i c a n d a r o m a t i c molecules, a n d by m i c r o b i a l s y n t h e s i s , a n d t h e s e m a t e r i a l s l a r g e l y c o n t r i b u t e to t h e p h y s i c o - c h e m i c a l s t a b i l i t y of t h e e c o s y t e m . I n o u r r e c e n t s t u d i e s we h a v e s h o w n t h a t b o t h living a n d d e a d S. alterniflora c o n t a i n h u m i c - l i k e c o n s t i t u e n t s w h i c h m a y b e c o m e i n c o r p o r a t e d i n t o t h e pool of h u m i c s u b s t a n c e s in s a l t m a r s h e s (Alberts et al., 1988; Filip et al., 1988).
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274 Different quantities of these humic-like plant constituents are released into sea water, a n d their characteristics resemble those of marine fulvic acids or marine humic acids (Filip and Alberts, 1988). The purpose of this study was to investigate whether there are humic-like substances in the S. alterniflora which remain in the plant tissue during long-term decay of the plant fragments in sea water, since they could contribute directly to the pool of humic material in the salt marsh sediment (mud). MATERIALS AND METHODS Sampling and preparation of the plant material, the preparation and use of inoculum, isolation of humic substances and analytical procedures were described in detail previously (Filip and Alberts, 1988; Filip et al., 1988). In brief, standing living and dead stems and leaves of S. alterniflora were collected from salt marshes of Sapelo Island, Georgia, U.S.A. For each experimental variant, 2 kg of the green plant material, and an equivalent weight of the dead plant debris, were allowed to decompose for 10 months in the dark at room temperature in flasks containing 71 of sea water. Prior to this, the flasks were sterilized by steam and they remained either sterile (controls) or were inoculated with a mixed population of epiphytic microorganisms associated with the living S. alterniflora (the inoculum to the fresh plant material) or associated with the dead S. alterniflora (the inoculum to the dead plant material). Other variants were inoculated with the natural mixed microbial population indigenous to the mud or with the microscopic fungi inhabiting the dead S. alterniflora stems, i.e. Leptosphaeria obiones or Phaeosphaeria typharum. In this way, five experimental variants were obtained for both the fresh and dead S. alterniflora plant material. After the 10 months, the plant material was dried at 60°C, finely milled, and portions (100 g) were extracted under N2 with 11 of a 0 . 1 M N a O H + 0.1MNa4P207 mixture ( l : l v / v ) for 24h. Humic substances were precipitated at pH 1, dialyzed against deionized water and freeze-dried. Their yield was estimated gravimetrically. Analyses for carbon, hydrogen and nitrogen were performed using a Perkin Elmer Analyzer Model 240C. Oxygen was determined by difference. Samples were heated overnight at 500°C to determine ash contents. The absorbance of the humic substances in the visible and ultraviolet range of light were measured after Chen et al. (1977) using a Hitachi 100-80 A recording spectrophotometer. For Fourier-transform infrared spectroscopy (FTIR), KI pellets containing 2% samples were examined in a Bruker 1FS 85 FTIR spectrophotometer. RESULTS As shown in Table 1, both the sterile controls and the inoculated variants yielded appreciable amounts of humic substances, but the quantity varied considerably. In general, the dead plant material yielded more humic
275 TABLE 1 Yields of humic substances from S. alterniflora plant material decomposingfor 10 months in sea water Sample and inoculum
Humic substance (dry weight) (mg/lO0g)
(%)
393.5 955.5 1054.1 581.3 581.9
100.0 242.8 268.0 147.7 147.8
580.9 1133.5 1166.5 568.6 721.3
100.0 195.1 200.8 101.3 124.2
S. alterniflora, fresh
Control Epiphytic microorganisms Mud microorganisms L. obiones P. typharum S. alterniflora, dead
Control Epiphytic microorganisms Mud microorganisms L. obiones P. typharum
substances t h a n the fresh plant material. The largest difference amounted to 48% and was recorded for the controls. The individual variants inoculated with different microorganisms did not display large differences among each other. Both fresh and dead plant material decomposing in the presence of mixed populations of microorganisms either from plant surfaces or the salt marsh mud yielded about twice as much humic substances as the controls. The mud microorganisms were the most effective in this respect, enhancing the yield of humic substances from fresh plant material by 168% in comparison with the control. The microscopic fungi L. obiones and P. t y p h a r u m were much less effective in yielding humic substances. L. obiones growing for 10 months on the dead plant material almost failed to increase the yield of humic substances in comparison with the control. Elemental compositions of the individual preparations of humic substances are shown in Table 2. The data for C, H and N are mean values from three estimations, with a standard deviation ranging from zero to 0.26%. The data show carbon and oxygen to be the most abundant elements in all preparations, followed by hydrogen and nitrogen. On average, the humic substances from the fresh plant material contained 4% more carbon but 5% less oxygen than those from the dead plant debris. The N contents were more similar. The individual atomic ratios for hydrogen, carbon and nitrogen mainly show similarities when compared with the values obtained for humic materials from the individual experimental variants. All the humic substances extracted from the S. alterniflora plant material show low ash contents, ranging from 1.28 to 2.39%. The absorption spectra of the humic substances in the visible region are shown in Figs 1-3. The preparations extracted from the individual variants of the fresh plant material gave spectra with an absorption shoulder at 670 nm,
276 TABLE 2 Chemical composition of humic substances from S. alterniflora plant material decomposing for 10 months in sea water; some atomic ratios and ash contents (elements in ash-free %) Sample and inoculum
C
N
H
O
H/C
O/C
N/C
Ash
56.11 58.49 57.17 56.72 57.05
2.45 3.05 2.59 2.55 2.16
6.19 6.87 6.01 6.22 5.99
35.25 31.59 34.23 34.51 34.80
1.32 1.41 1.26 1.32 1.26
0.47 0.40 0.45 0.46 0.46
0.04 0.05 0.04 0.04 0.03
2.39 1.79 1.75 1.28 1.43
52.25 52.47 53.39 53.80 52.89
3.12 2.73 2.30 2.33 2.82
5.33 4.90 5.15 5.65 5.50
39.30 39.90 39.16 38.22 38.79
1.23 1.13 1.16 1.26 1.25
0.57 0.57 0.55 0.53. 0.55
0.05 0.04 0.04 0.04 0.05
2.12 2.16 1.97 1.65 1.60
(%)
S. alterniflora, fresh Control Epiphytic microorganisms Mud microorganisms
L. obiones P. typharum S. alterniflora, dead Control Epiphytic microorganisms Mud microorganisms
L. obiones P. typharum
100
///1-A Ill D E
C4 BI LIJ 0 Z
~: 5o 0 m
0
i
400
500
6 0
700
800
WAVELENGTH, nm
Fig. 1. Visible spectra of humic substances from fresh S. alterniflora decomposing for 10 months in sea water: (A) control; (B) from the variant inoculated with a mixed population of epiphytic microorganisms from fresh plants; (C) from the variant inoculated with a mixed population of mud microorganisms; (D) from the variant inoculated with L. obiones; (E) from the variant inoculated with P. typharum.
277 I00
I
uJ o z
~:
50
0 m
0
i
400
500
i
t
600 700 WAVELENGTH, nm
800
Fig. 2. Visible spectra of humic substances from dead S. alterniflora decomposing for 10 months in sea water: (F) control; (G) from the v a r i a n t inoculated with a mixed population of epiphytic microorganisms from dead plants; (H) from the v a r i a n t inoculated with a mixed population of mud microorganisms; (I) from the v a r i a n t inoculated with L. obiones; (J) from the v a r i a n t inoculated with P. typharum. lO0
LU
o z
0
i
400
500
t
t
600 700 WAVELENGTH, nm
800
Fig. 3. Comparison o f visible spectra o f hulllic substances f r o m fresh, non-incubated S. alterni~Zore,
those from the v a r i a n t (G) (see Fig. 2), and of a humic acid from the salt marsh mud.
278
the intensity of which was most strongly expressed in the humic substance from the control. The spectrum of the humic substance from the variant inoculated with a mixed population of epiphytic microorganisms was featureless. Featureless spectra were also obtained from all humic preparations from the dead plant material (Fig. 2). In Fig. 3 a comparison has been made between humic substances from non-incubated fresh plant material, from salt marsh mud and from dead S. alterniflora inoculated with the epiphytic microorganisms. These visible spectra clearly indicate that the optical density of the latter preparation is almost identical to that of humic substances from mud. A weak absorption shoulder at 670 nm in the spectrum of the mud humic substance may be attributed to constituents of the humic substances from the fresh S. alter-
niflora. The UV spectra of all humic preparations exhibit absorption maxima at 275 nm and weak shoulders at 320 urn. These are typically shown in Fig. 4. From this figure, one can recognize that the humic substance from salt marsh mud also weakly absorbs at the same wavelength. In Table 3 the extinction coefficients measured at 465 and 665 nm, and also the values of the E4/E6 ratio, are given. The data are in the range 3.29-7.22, which is typical for humic and fulvic acids. Lower values of the E4/E6 ratio are generally associated with humic substances recovered from plant material inoculated with the mixed microbial populations. The FTIR spectra of the humic substances are shown in Figs 5 and 6, and 100
B
G
B
~J o
z
~ 5o 0 m
0 200
3(]0 WAVELENGTH, nm
400
Fig. 4. Comparison of UV spectra from the v a r i a n t s (B) and (G) (see Figs 1 and 2) and the humic acid from the salt marsh mud.
279 TABLE 3 Extinction coefficients of humic substances at 465 and 665 nm, and the values of E4/E6 ratios Humic substances from (sample and inoculum)
E465
E665
E4/E6
0.450 0.435 0.430 0.445 0.361
0.111 0.132 0.093 0.064 0.050
4.05 3.29 4.62 6.95 7.22
0.368 0.601 0.575 0.402 0.455 0.555
0.057 0.134 0.115 0.068 0.074 0.110
6.46 4.49 5.00 5.91 6.15 5.05
S. alterniflora, fresh Control Epiphytic microorganisms Mud microorganisms
L. obiones P. typharum S. alterniflora, dead Control Epiphytic microorganisms Mud microorganisms
L. obiones P. typharum (Mud)
A Z
,
4000
,
,
,
3200
,
,
,
,
2400
,
.
.
.
.
.
.
.
.
1800 1400 WAVENUMBERS CM -1
.
.
.
.
1200 1000
.
.
.
800
.
600
400
Fig. 5. FTIR spectra of the humic substances from fresh S. alterniflora decomposing for 10 months in sea water. (For symbols, see Fig. 1.)
280
F
.._J
.-...j
H
....J
I
__J
4000
3200
2400
1800 1400 1200 WAVENUMBERS C M - 1
1000
800
600
400
Fig. 6. FTIR spectra of the humic substances from dead S. alterniflora decomposing for 10 months in sea water. (For symbols, see Fig. 2).
TABLE 4 Infrared absorption bands of humic substances from long-term decomposition of S. alterniflora in sea water (Assignments according to Orlov et al., 1972; Stevenson, 1982; McCarthy and Rice, 1985, and others.) Band (cm -1)
Possible assignment
3385-3360 2920 2850 1715-1700 1655 1635 1605
H-bonded OH groups and partly NH groups Aliphatic C-H stretching Aliphatic C-H stretching C = O stretching of COOH and ketonic C = O C = O stretching, amide I C = O stretching (amide) COH group, CH2-, CH3-stretching, aromatic ring stretching C = C aromatic ring stretching (amide II) C-H deformation of CH 2 or CH3 groups C = C aromatic ring stretching -CH3 stretching, OH in alcohols and phenols C-O stretching and OH deformation of COOH; C-N of amide III C-O stretching of carbohydrates C-O stretching (polysaccharides, aromatics) C-H bond (aromatics)
1515 1463 1422 1340-1330 1266-1225 1126 1034 835
281 possible assignments of the individual absorption bands are listed in Table 4. Since the humic substances extracted from the plant materials inoculated with either L. obiones or P. typharum exhibited almost identical spectra, the FTIR spectra from the P. typharum cultures have been omitted in these figures. The major feature of the FTIR spectra of all humic substances under examination is the OH-stretching bands in the 3300 cm 1 region. Their intensity, however, is clearly diminished in preparations from live plant cultures inoculated with the mixed populations of microorganisms (Fig. 5B,C). The absorption by aliphatic C-H bonds of methyl and/or methylene groups at 2920 and 2850 cm- 1 is strongly expressed in the spectra of humic substances from the fresh plant material but considerably weaker in those associated with the dead plant material. The C = O bonding of carboxyl and carbonyl groups produce bands in the spectra of some preparations from the fresh plants, and appear only as shoulders in all other spectra. An amide I absorption band appears in the spectra of all humic substances. In some spectra from dead plant material, it is weaker and split (1635 cm- 1 absorption in Fig. 6H,I). The absorption bands at 1605 and 1515 cm -1 may indicate C = C aromatic ring stretching. However, with respect to the character of plant-associated humic material, and perhaps microbial by-products, CH 2- and CH3-stretching as well as the amide II absorption should also be considered. Also, the presence of the sharp absorption band at 1463cm -1 indicates C-H deformation of methyl and methoxyl groups. Nevertheless, aromatic ring-stretching peaks at 1422 cm 1 occur in all spectra of humic preparations under examination. A weak absorption appears at 1340-1330 cm-1, which may indicate phenolic OH groups. In the 1266-1225 cm -1 region, double absorption bands appear, both peaks of which can be attributed to the C-O stretching vibrations of COOH and/or of C-N groups in amide III. Humic substances from the inoculated fresh plant material all show a strong C-O stretching absorption of carbohydrates at 1126 and 1034 cm 1, and similar features can be observed in spectra of the dead plant material. Silicate impurities do not seem to contribute to these absorptions, due to the low ash contents of all humic preparations examined. C-H bending absorptions of aromatics appear at 835 cm-1 in all spectra of the S. alternifloraderived humic substances. DISCUSSION Because of extensive biochemical and microbial transformations during early decomposition, humic compounds represent that part of organic matter from which all substances considered to be readily available sources of energy have been exhausted (Rashid, 1985). Thus, if the organic substances isolated by Alberts et al. (1988) and Filip et al. (1988) from living and dead S. alterniflora are considered to be humic compounds, they must also be able to persist during the decomposition of fresh plant material and dead plant debris in sea water. The results of this study show this to be true, both for the plant materials kept for 10 months under sterile conditions and those incubated after inoculation
282 with different microorganisms. Whereas some amounts of humic-like substances have been shown to be released into sea water (Filip and Alberts, 1988), appreciable quantities remain bound in the plant tissues. Epiphytic microorganisms and microorganisms indigenous to mud enhanced the yield of humic substances up to 168% from the fresh plant material and up to 100% from the dead debris. From the carbon content of S. alterniflora (fresh 40.7%; dead, 52.0%; Filip and Alberts, 1988) and from the data in Tables 1 and 2, one can calculate that a maximum of 1.48% of the C of the incubated fresh plant material and 1.20% of the C of the incubated dead plant material was present as humic substances. Since the humic substances content of living S. alterniflora was calculated to be 0.64% of the total plant carbon (Filip et al., 1988), one has to assume an additional humification of the plant biomass by the mixed microbial populations in sea water during the incubation period. For P. typharum growing on the fresh plant material submerged in sea water, 0.82% of the total plant carbon was found in the humic substances. The data for L. obiones were similar, indicating a lower humification activity of these fungi in the submerged plant material. In comparison with the elemental composition of humic substances extracted from fresh samples of living S. alterniflora, which was reported to be 46% C and 5.8% N by Filip et al. (1988), humic substances examined in this study exhibited a higher C content (56.11-58.49%) and a lower N content (2.16-3.05%). This may be due to microbial utilization of simple structures of the plant humic substance during the long-term incubation. The remaining humic compounds may then be more resistant to further decomposition. These differences are not strongly expressed with the humic substances from the dead plant debris. Generally, the elemental data of all humic substances recovered in this experiment correspond well with those reported by others for aquatic or sedimentary humic acids (Ishiwatary, 1975; Stevenson, 1982; Rashid, 1985; Steelink, 1985). The atomic ratio of hydrogen to carbon is related to the percentage saturation of the carbon atoms, and is therefore important for aquatic humic substances. According to Thurman (1985), an H/C ratio of 2:1 indicates that the carbon atoms are aliphatic in character, whereas a ratio of 1:1 indicates aromatic structures. Since the H/C ratios of all the preparations examined in this study are in the range 1.13-1.41, one can assume the presence of aromatic structures. Ertel and Hedges (1985) listed similar values for humic substances obtained from fresh and degraded plant tissues. The O/C ratios give values between 0.40 and 0.57, and the values of N/C ratios are between 0.03 and 0.05, i.e. in a range typical for soil and aquatic humic acids, according to Steelink (1985). The extinction curves recorded with visible light mainly exhibit features which are typical for humic acids (Figs 1 and 2). The small maxima (or shoulders) at 670nm which appear in some preparations (Fig. 1) can be attributed to plant porphyrines, but they are also typical of marine humic acid,
283 according to Ertel and Hedges (1983). The curves in Fig. 3 demonstrate that some humic preparations from this experiment exhibit an extinction curve which is almost identical to that of the mud humic acid, and that the 670 nm extinction of the mud humic acid may originate from humic compounds related to the freshly collected S. alterniflora. The E4]E6 ratios given in Table 3 correspond well with the values found for sedimentary humic acids by Kalinowski and Blondeau (1988). The similarity between the mud humic acid and humic substances extracted from either fresh or dead S. alterniflora decomposing in sea water containing the mixed population of salt marsh indigenous microorganisms can be seen clearly also from Fig. 4. There are common spectral features, shown as absorption maxima or shoulders at 275-280 and 320 nm. Filip et al. (1976) found similar absorption maxima for humic-like fungal melanins, whereas Goldschmid (1971) reported absorbance in the same regions for phenolic structures of grass lignin. FTIR spectra of the humic substances from S. alterniflora show all the major features typical for humic acids or for model polymers isolated from either terrestrial or marine environments and which were reported by Filip et al. (1974), Stevenson (1982), MacCarthy and Rice (1985), and Thurman (1985). However, the interpretation of amide bands at 1655, 1515 and 1266-1225 cm -1 should be made with care because of the low N contents of the humic preparations tested. In these particular positions, C = O stretching and C -- C aromatic ring stretching must also be considered. The need for caution in interpreting these bands is further emphasized by the fact that it may be calculated for marsh sediment fulvic and hnmic acids that 35 and 20% of the nitrogen, respectively, is in the form of amino acids (Alberts and Filip, 1988). Thus, of the already low nitrogen content of these humic substances, a significant portion may comprise amino acid nitrogen, further reducing the potential amount of amide-bound nitrogen in the compounds. Salt marshes have long been recognized as being among the most productive ecosystems in the world (Odum, 1959). According to McLusky (1981), who referred to several individual authors, the production of the above-ground material of S. alterniflora may be up to 3300 g dry weight per square metre per year. This copious biomass is regularly utilized by numerous species of bacteria and fungi under both aerobic and anaerobic conditions (Gessner and Goos, 1973; Haines and Hanson, 1979). The degradation processes naturally result in a slower or faster mineralization of the plant biomass and enhancement of microbial numbers (Newell et al., 1985; Chrzanowski and Spurrier, 1987). However, our results show that there is also organic matter in the tissue of S. alterniflora which apparently resists degradation. This organic matter remains stable during long-term decomposition of plant fragments in sea water, and shows characteristics which are typical for sedimentary humic acids. The amounts of these humic substances increased substantially in the experimental variants inoculated with mixed populations of microorganisms indigenous to salt marshes.
284 T h e s e findings a g r e e well w i t h the thesis f o r m u l a t e d r e c e n t l y by H a t c h e r a n d S p i k e r (1988), w h i c h claims t h a t r e s i s t a n t f r a c t i o n s of fresh p l a n t m a t e r i a l are c o m p a r a b l e to h u m i c s u b s t a n c e s w i t h i n t h e c o n t e x t of t h e d e g r a d a t i v e s c h e m e for h u m i f i c a t i o n , b e c a u s e t h e y m a y b e c o m e e n r i c h e d d u r i n g d e g r a d a tion. F u r t h e r m o r e , o u r r e s u l t s b r i n g e x p e r i m e n t a l s u p p o r t to t h e a s s u m p t i o n m a d e by C r a f t et al. (1988), u s i n g s t a b l e i s o t o p e s of c a r b o n and n i t r o g e n . A c c o r d i n g to t h e isotopic c o m p o s i t i o n of m a r s h soils, t h e a u t h o r s deduced t h a t t h e m a r s h e m e r g e n t v e g e t a t i o n a p p e a r s to be t h e p r i n c i p a l s o u r c e of o r g a n i c m a t t e r in soils of t h e s a l t m a r s h e s . F r o m o u r results, a n d w i t h r e s p e c t to the a n n u a l yield of S. alterniflora d r y m a t t e r in a s a l t m a r s h as g i v e n by M c L u s k y (1981), one c a n c a l c u l a t e a p o t e n t i a l a n n u a l i n p u t of a b o u t 3 3 0 k g of fresh S p a r t i n a - r e l a t e d h u m i c s u b s t a n c e s p e r h e c t a r e . T h e r e is no d o u b t t h a t s u c h a n i n p u t m a y s u b s t a n t i a l l y c o n t r i b u t e to t h e l o n g - t e r m p h y s i c o - c h e m i c a l a n d b i o l o g i c a l s t a b i l i t i e s of e s t u a r i n e s a l t m a r s h e s . ACKNOWLEDGEMENTS T h e a u t h o r s t h a n k Dr H.P. R e i s e n a u e r of t h e U n i v e r s i t y of G i e s s e n for the F T I R m e a s u r e m e n t s a n d Dr R. S m e d - H i l d m a n n ( L a n g e n ) for s u p e r v i s i n g the i s o l a t i o n of h u m i c s u b s t a n c e s . T h e y also a c k n o w l e d g e t h e skilled t e c h n i c a l a s s i s t a n c e of Miss M. P r i c e (Sapelo I s l a n d ) a n d Miss A. H e e r (Langen). T h i s r e s e a r c h w a s funded, in p a r t , by the S a p e l o I s l a n d R e s e a r c h F o u n d a t i o n a n d t h e D e u t s c h e F o r s c h u n g s g e m e i n s c h a f t , Bonn. This p a p e r is a j o i n t c o n t r i b u tion of t h e U G A M a r i n e I n s t i t u t e (No. 625) a n d the I n s t i t u t fiir Wasser-, Bodenu n d L u f t h y g i e n e des B u n d e s g e s u n d h e i t s a m t e s . REFERENCES Alberts, J.J. and Z. Filip, 1988. Sources and characteristics of fulvic and humic acids from a salt marsh estuary. Sci. Total Environ., in press. Alberts, J.J., Z. Filip, M.T. Price, D.J. Williams and M.C. Williams, 1988. Elemental composition, stable carbon isotope ratios and spectrophotometric properties of humic substances occurring in a salt marsh estuary. Org. Geochem., 12: 455-467. Chen, Y., N. Senesi and M. Schnitzer, 1977. Information provided on humic substances by EJE 8 ratios. Soil Sci. Soc. Am., 41: 352-358. Chrzanowski, T.H. and J.D. Spurrier, 1987. Exchange of microbial biomass between a Spartina alterniflora marsh and the adjacent tidal creek. Estuaries, 10: 118-125. Craft, C.B., S.W. Broome, E.D. Seneca and W.J. Showers, 1988. Estimating sources of soil organic matter in natural and transplanted estuarine marshes using stable isotopes of carbon and nitrogen. Estuarine Coastal Shelf Sci., 26: 633-641. Ertel, J.R. and J.I. Hedges, 1983. Bulk chemical and spectroscopic properties of marine and terrestrial humic acids, melanoidins and catechol-based synthetic polymers. In: R.F. Christman and E.T. Gjessing (Eds), Aquatic and Terrestrial Humic Materials. Ann Arbor Science, Ann Arbor, Michigan, pp. 143-163. Ertel, J.R. and J.I. Hedges, 1985. Sources of sedimentary humic substances: vascular plant debris. Geochim. Cosmochim. Acta, 48: 2065-2074. Fallon, R.D. and J.K. Pfaender, 1976. Carbon metabolism in model microbial system from a temperate salt marsh. Appl. Environ. Microbiol., 31: 959-968.
285 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., K. Haider, H. Beutelspacher and J.P. Martin, 1974. Comparison of IR°spectra from melanins of microscopic soil fungi, humic acids and model phenol polymers. Geoderma, 11: 37-52. Filip, Z., J. Semotan and M. Kutilek, 1976. Thermal and spectrophotometric analysis of some fungal melanins and soil humic compounds. Geoderma, 15: 131-142. 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. Gessner, R.V. and R.D. Goos, 1973. Fungi from decomposing Spartina alterniflora. Can. J. Bot., 51: 51-55. Goldschmid, O., 1971. Ultraviolet spectra. In: K.V. Sarkanen and C.H. Ludwig (Eds), Lignins: Occurrence, Formation, Structure and Reactions, John Wiley and Sons, New York, pp. 241 266. Haines, E.B. and R.B. Hanson, 1979. Experimental degradation of detritus made from the salt marsh plants Spartina alterniflora Loisel., Salicornia virginica L., and Junctus roemerianus Scheele. J. Exp. Mar. Biol. Ecol., 40: 27-40. Hatcher, P.G. and E.C. Spiker, 1988. Selective degradation of plant biomolecules. In: F.H. Frimmel and R.F. Christman (Eds), Humic Substances and Their Role in the Environment. John Wiley and Sons, Chichester, pp. 59--74. Ishiwatari, R., 1975. Chemical nature of sedimentary humic acids. In: Proc. Int. Meet. Humic Substances, Nieuwersluis, 1972. Pudoc, Wageningen, pp. 87-107. Kalinowski, E. and R. Blondeau, 1988. Characterization of sedimentary humic acids fractionated by hydrophobic interaction chromatography. Mar. Chem., 24: 29-37. MacCarthy, P. and J.A. Rice, 1985. Spectroscopic methods (other than NMR) for determining functionality in humic substances. In: G.R. Aiken, D.M. McKnight, R.L. Wershaw and P. MacCarthy (Eds), Humic Substances in Soil, Sediment, and Water. John Wiley and Sons, New York, pp. 527-559. Mann, K.H., 1988. Towards predictive models for coastal marine ecosystems. In: L.R. Pomeroy and J.J. Alberts (Eds), Concepts of Ecosystem Ecology. Springer-Verlag, New York, pp. 291-316. McLusky, D.S., 1981. The Estuarine Ecosystem. John Wiley and Sons, New York, 150 pp. Newell, S.Y., R.D. Fallon, R.M. Cal Rodrigues and L.C. Groene, 1985. Influence of rain, tidal wetting and relative humidity on release of carbon dioxide by standing-dead salt-marsh plants. Oecologia (Berlin), 68: 73-79. Odum, E.P., 1959. Fundamentals of Ecology. W.B. Saunders, New York, 574pp. Orlov, D.C., Ju. A. Shulman and A.A. Jukhnin, 1972. Infrared spectra of soil humic acids and a table of wavenumbers. In: M.M. Kononova (Ed.), Organic Substances of Virgin and Cultivated Soils. Nauka, Moscow, pp. 245-256 (in Russian). 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. Pomeroy, L.R., W.M. Darley, E.L. Dunn, J.L. Gallager, E.B. Haines and D.M. Whitney, 1981. Primary production. In: L.R. Pomeroy and R.G. Wiegert (Eds), The Ecology of Salt Marsh. Springer-Verlag, New York, pp. 71-74. Rashid, M.A., 1985. Geochemistry of Marine Humic Compounds. Springer-Verlag, New York, 300 pp. Steelink, C., 1985. Implications of elemental characteristics of humic substances. In: G.R. Aiken, D.M. McKnight, R.L. Weshaw and P. MacCarthy (Eds), Humic Substances in Soil, Sediment, and Water. John Wiley and Sons, New York, pp. 457-476. Stevenson, F.J., 1982. Humus Chemistry. John Wiley and Sons, New York, 443 pp. Teal, J.M., 1962. Energy flow in the salt marsh ecosystem of Georgia. Ecology, 43: 614-624. Thurman, E.M., 1985. Organic Geochemistry of Natural Waters. Martinus Nijhoff/Dr. W. Junk Publishers, Dodrecht, 497 pp.
273
HUMIC SUBSTANCES ISOLATED FROM SPARTINA ALTERNIFLORA (LOISEL.) FOLLOWING LONG-TERM DECOMPOSITION IN SEA WATER
Z. FILIP Institut fiir Wasser-, Boden- und Lufthygiene des Bundesgesundheitsamtes, Aussenstelle Langen, Paul.Ehrlich-Strasse 29, D-6070 Langen (Federal Republic of Germany) J.J. ALBERTS The University of Georgia, Marine Institute, Sapelo Island, GA 31327 (U.S.A.) (Received December 1st, 1968; accepted January 12th, 1989)
ABSTRACT Fragments of fresh or dead Spartina alterniflora which were decomposed for 10 months in sea water yielded appreciable amounts of humic substances when extracted with alkali under nitrogen. The plant materials inoculated with mixed populations of epiphytic or mud microorganisms indigenous to the salt marsh yielded up to 168% more humic substances than controls. The results of elemental analyses, including atomic ratios for some elements, and also UV and Fouriertransform infrared (FrIR) spectroscopy investigations, indicate a great similarity between humic substances from the decomposing S. alterniflora and salt marsh humic acid. Calculations based on the experimental data indicate a potential annual input of ~ 330 kg of fresh Spartina-related humic substances per hectare of salt marsh. INTRODUCTION On s e d i m e n t i n g s h o r e l i n e s in t e m p e r a t e l a t i t u d e s , l a r g e a r e a s of t h e u p p e r i n t e r t i d a l zone a r e o f t e n d o m i n a t e d by S p a r t i n a m a r s h e s (Mann, 1988). Also, t h e p r i m a r y p r o d u c t i o n o f b i o m a s s o c c u r r i n g in t h e s a l t m a r s h e s of t h e A t l a n t i c a n d G u l f c o a s t s of t h e U.S.A. t a k e s p l a c e m a i n l y in t h e v a s t s t a n d s of t h e s m o o t h c o r d g r a s s S p a r t i n a alterniflora (Loisel.). T h e losses of dissolved o r g a n i c m a t t e r f r o m living s h o o t s of S. alterniflora d u r i n g t i d a l s u b m e r g e n c e in t h e s a l t m a r s h e s , a n d f r o m detritus, i.e. d e a d p l a n t f r a g m e n t s a n d a s s o c i a t e d m i c r o o r ganisms which are almost permanently submerged, represent an important i n p u t of n u t r i e n t s i n t o e s t u a r i n e food webs (Teal, 1962; H a i n e s a n d H a n s o n , 1979; P o m e r o y et al., 1981; P a k u l s k i , 1986). S i m u l t a n e o u s l y , n e w f o r m s of r e f r a c t o r y o r g a n i c m a t t e r , s u c h as h u m i c s u b s t a n c e s , c a n be f o r m e d by a b i o t i c p o l y m e r i z a t i o n o f simple a l i p h a t i c a n d a r o m a t i c molecules, a n d by m i c r o b i a l s y n t h e s i s , a n d t h e s e m a t e r i a l s l a r g e l y c o n t r i b u t e to t h e p h y s i c o - c h e m i c a l s t a b i l i t y of t h e e c o s y t e m . I n o u r r e c e n t s t u d i e s we h a v e s h o w n t h a t b o t h living a n d d e a d S. alterniflora c o n t a i n h u m i c - l i k e c o n s t i t u e n t s w h i c h m a y b e c o m e i n c o r p o r a t e d i n t o t h e pool of h u m i c s u b s t a n c e s in s a l t m a r s h e s (Alberts et al., 1988; Filip et al., 1988).
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274 Different quantities of these humic-like plant constituents are released into sea water, a n d their characteristics resemble those of marine fulvic acids or marine humic acids (Filip and Alberts, 1988). The purpose of this study was to investigate whether there are humic-like substances in the S. alterniflora which remain in the plant tissue during long-term decay of the plant fragments in sea water, since they could contribute directly to the pool of humic material in the salt marsh sediment (mud). MATERIALS AND METHODS Sampling and preparation of the plant material, the preparation and use of inoculum, isolation of humic substances and analytical procedures were described in detail previously (Filip and Alberts, 1988; Filip et al., 1988). In brief, standing living and dead stems and leaves of S. alterniflora were collected from salt marshes of Sapelo Island, Georgia, U.S.A. For each experimental variant, 2 kg of the green plant material, and an equivalent weight of the dead plant debris, were allowed to decompose for 10 months in the dark at room temperature in flasks containing 71 of sea water. Prior to this, the flasks were sterilized by steam and they remained either sterile (controls) or were inoculated with a mixed population of epiphytic microorganisms associated with the living S. alterniflora (the inoculum to the fresh plant material) or associated with the dead S. alterniflora (the inoculum to the dead plant material). Other variants were inoculated with the natural mixed microbial population indigenous to the mud or with the microscopic fungi inhabiting the dead S. alterniflora stems, i.e. Leptosphaeria obiones or Phaeosphaeria typharum. In this way, five experimental variants were obtained for both the fresh and dead S. alterniflora plant material. After the 10 months, the plant material was dried at 60°C, finely milled, and portions (100 g) were extracted under N2 with 11 of a 0 . 1 M N a O H + 0.1MNa4P207 mixture ( l : l v / v ) for 24h. Humic substances were precipitated at pH 1, dialyzed against deionized water and freeze-dried. Their yield was estimated gravimetrically. Analyses for carbon, hydrogen and nitrogen were performed using a Perkin Elmer Analyzer Model 240C. Oxygen was determined by difference. Samples were heated overnight at 500°C to determine ash contents. The absorbance of the humic substances in the visible and ultraviolet range of light were measured after Chen et al. (1977) using a Hitachi 100-80 A recording spectrophotometer. For Fourier-transform infrared spectroscopy (FTIR), KI pellets containing 2% samples were examined in a Bruker 1FS 85 FTIR spectrophotometer. RESULTS As shown in Table 1, both the sterile controls and the inoculated variants yielded appreciable amounts of humic substances, but the quantity varied considerably. In general, the dead plant material yielded more humic
275 TABLE 1 Yields of humic substances from S. alterniflora plant material decomposingfor 10 months in sea water Sample and inoculum
Humic substance (dry weight) (mg/lO0g)
(%)
393.5 955.5 1054.1 581.3 581.9
100.0 242.8 268.0 147.7 147.8
580.9 1133.5 1166.5 568.6 721.3
100.0 195.1 200.8 101.3 124.2
S. alterniflora, fresh
Control Epiphytic microorganisms Mud microorganisms L. obiones P. typharum S. alterniflora, dead
Control Epiphytic microorganisms Mud microorganisms L. obiones P. typharum
substances t h a n the fresh plant material. The largest difference amounted to 48% and was recorded for the controls. The individual variants inoculated with different microorganisms did not display large differences among each other. Both fresh and dead plant material decomposing in the presence of mixed populations of microorganisms either from plant surfaces or the salt marsh mud yielded about twice as much humic substances as the controls. The mud microorganisms were the most effective in this respect, enhancing the yield of humic substances from fresh plant material by 168% in comparison with the control. The microscopic fungi L. obiones and P. t y p h a r u m were much less effective in yielding humic substances. L. obiones growing for 10 months on the dead plant material almost failed to increase the yield of humic substances in comparison with the control. Elemental compositions of the individual preparations of humic substances are shown in Table 2. The data for C, H and N are mean values from three estimations, with a standard deviation ranging from zero to 0.26%. The data show carbon and oxygen to be the most abundant elements in all preparations, followed by hydrogen and nitrogen. On average, the humic substances from the fresh plant material contained 4% more carbon but 5% less oxygen than those from the dead plant debris. The N contents were more similar. The individual atomic ratios for hydrogen, carbon and nitrogen mainly show similarities when compared with the values obtained for humic materials from the individual experimental variants. All the humic substances extracted from the S. alterniflora plant material show low ash contents, ranging from 1.28 to 2.39%. The absorption spectra of the humic substances in the visible region are shown in Figs 1-3. The preparations extracted from the individual variants of the fresh plant material gave spectra with an absorption shoulder at 670 nm,
276 TABLE 2 Chemical composition of humic substances from S. alterniflora plant material decomposing for 10 months in sea water; some atomic ratios and ash contents (elements in ash-free %) Sample and inoculum
C
N
H
O
H/C
O/C
N/C
Ash
56.11 58.49 57.17 56.72 57.05
2.45 3.05 2.59 2.55 2.16
6.19 6.87 6.01 6.22 5.99
35.25 31.59 34.23 34.51 34.80
1.32 1.41 1.26 1.32 1.26
0.47 0.40 0.45 0.46 0.46
0.04 0.05 0.04 0.04 0.03
2.39 1.79 1.75 1.28 1.43
52.25 52.47 53.39 53.80 52.89
3.12 2.73 2.30 2.33 2.82
5.33 4.90 5.15 5.65 5.50
39.30 39.90 39.16 38.22 38.79
1.23 1.13 1.16 1.26 1.25
0.57 0.57 0.55 0.53. 0.55
0.05 0.04 0.04 0.04 0.05
2.12 2.16 1.97 1.65 1.60
(%)
S. alterniflora, fresh Control Epiphytic microorganisms Mud microorganisms
L. obiones P. typharum S. alterniflora, dead Control Epiphytic microorganisms Mud microorganisms
L. obiones P. typharum
100
///1-A Ill D E
C4 BI LIJ 0 Z
~: 5o 0 m
0
i
400
500
6 0
700
800
WAVELENGTH, nm
Fig. 1. Visible spectra of humic substances from fresh S. alterniflora decomposing for 10 months in sea water: (A) control; (B) from the variant inoculated with a mixed population of epiphytic microorganisms from fresh plants; (C) from the variant inoculated with a mixed population of mud microorganisms; (D) from the variant inoculated with L. obiones; (E) from the variant inoculated with P. typharum.
277 I00
I
uJ o z
~:
50
0 m
0
i
400
500
i
t
600 700 WAVELENGTH, nm
800
Fig. 2. Visible spectra of humic substances from dead S. alterniflora decomposing for 10 months in sea water: (F) control; (G) from the v a r i a n t inoculated with a mixed population of epiphytic microorganisms from dead plants; (H) from the v a r i a n t inoculated with a mixed population of mud microorganisms; (I) from the v a r i a n t inoculated with L. obiones; (J) from the v a r i a n t inoculated with P. typharum. lO0
LU
o z
0
i
400
500
t
t
600 700 WAVELENGTH, nm
800
Fig. 3. Comparison o f visible spectra o f hulllic substances f r o m fresh, non-incubated S. alterni~Zore,
those from the v a r i a n t (G) (see Fig. 2), and of a humic acid from the salt marsh mud.
278
the intensity of which was most strongly expressed in the humic substance from the control. The spectrum of the humic substance from the variant inoculated with a mixed population of epiphytic microorganisms was featureless. Featureless spectra were also obtained from all humic preparations from the dead plant material (Fig. 2). In Fig. 3 a comparison has been made between humic substances from non-incubated fresh plant material, from salt marsh mud and from dead S. alterniflora inoculated with the epiphytic microorganisms. These visible spectra clearly indicate that the optical density of the latter preparation is almost identical to that of humic substances from mud. A weak absorption shoulder at 670 nm in the spectrum of the mud humic substance may be attributed to constituents of the humic substances from the fresh S. alter-
niflora. The UV spectra of all humic preparations exhibit absorption maxima at 275 nm and weak shoulders at 320 urn. These are typically shown in Fig. 4. From this figure, one can recognize that the humic substance from salt marsh mud also weakly absorbs at the same wavelength. In Table 3 the extinction coefficients measured at 465 and 665 nm, and also the values of the E4/E6 ratio, are given. The data are in the range 3.29-7.22, which is typical for humic and fulvic acids. Lower values of the E4/E6 ratio are generally associated with humic substances recovered from plant material inoculated with the mixed microbial populations. The FTIR spectra of the humic substances are shown in Figs 5 and 6, and 100
B
G
B
~J o
z
~ 5o 0 m
0 200
3(]0 WAVELENGTH, nm
400
Fig. 4. Comparison of UV spectra from the v a r i a n t s (B) and (G) (see Figs 1 and 2) and the humic acid from the salt marsh mud.
279 TABLE 3 Extinction coefficients of humic substances at 465 and 665 nm, and the values of E4/E6 ratios Humic substances from (sample and inoculum)
E465
E665
E4/E6
0.450 0.435 0.430 0.445 0.361
0.111 0.132 0.093 0.064 0.050
4.05 3.29 4.62 6.95 7.22
0.368 0.601 0.575 0.402 0.455 0.555
0.057 0.134 0.115 0.068 0.074 0.110
6.46 4.49 5.00 5.91 6.15 5.05
S. alterniflora, fresh Control Epiphytic microorganisms Mud microorganisms
L. obiones P. typharum S. alterniflora, dead Control Epiphytic microorganisms Mud microorganisms
L. obiones P. typharum (Mud)
A Z
,
4000
,
,
,
3200
,
,
,
,
2400
,
.
.
.
.
.
.
.
.
1800 1400 WAVENUMBERS CM -1
.
.
.
.
1200 1000
.
.
.
800
.
600
400
Fig. 5. FTIR spectra of the humic substances from fresh S. alterniflora decomposing for 10 months in sea water. (For symbols, see Fig. 1.)
280
F
.._J
.-...j
H
....J
I
__J
4000
3200
2400
1800 1400 1200 WAVENUMBERS C M - 1
1000
800
600
400
Fig. 6. FTIR spectra of the humic substances from dead S. alterniflora decomposing for 10 months in sea water. (For symbols, see Fig. 2).
TABLE 4 Infrared absorption bands of humic substances from long-term decomposition of S. alterniflora in sea water (Assignments according to Orlov et al., 1972; Stevenson, 1982; McCarthy and Rice, 1985, and others.) Band (cm -1)
Possible assignment
3385-3360 2920 2850 1715-1700 1655 1635 1605
H-bonded OH groups and partly NH groups Aliphatic C-H stretching Aliphatic C-H stretching C = O stretching of COOH and ketonic C = O C = O stretching, amide I C = O stretching (amide) COH group, CH2-, CH3-stretching, aromatic ring stretching C = C aromatic ring stretching (amide II) C-H deformation of CH 2 or CH3 groups C = C aromatic ring stretching -CH3 stretching, OH in alcohols and phenols C-O stretching and OH deformation of COOH; C-N of amide III C-O stretching of carbohydrates C-O stretching (polysaccharides, aromatics) C-H bond (aromatics)
1515 1463 1422 1340-1330 1266-1225 1126 1034 835
281 possible assignments of the individual absorption bands are listed in Table 4. Since the humic substances extracted from the plant materials inoculated with either L. obiones or P. typharum exhibited almost identical spectra, the FTIR spectra from the P. typharum cultures have been omitted in these figures. The major feature of the FTIR spectra of all humic substances under examination is the OH-stretching bands in the 3300 cm 1 region. Their intensity, however, is clearly diminished in preparations from live plant cultures inoculated with the mixed populations of microorganisms (Fig. 5B,C). The absorption by aliphatic C-H bonds of methyl and/or methylene groups at 2920 and 2850 cm- 1 is strongly expressed in the spectra of humic substances from the fresh plant material but considerably weaker in those associated with the dead plant material. The C = O bonding of carboxyl and carbonyl groups produce bands in the spectra of some preparations from the fresh plants, and appear only as shoulders in all other spectra. An amide I absorption band appears in the spectra of all humic substances. In some spectra from dead plant material, it is weaker and split (1635 cm- 1 absorption in Fig. 6H,I). The absorption bands at 1605 and 1515 cm -1 may indicate C = C aromatic ring stretching. However, with respect to the character of plant-associated humic material, and perhaps microbial by-products, CH 2- and CH3-stretching as well as the amide II absorption should also be considered. Also, the presence of the sharp absorption band at 1463cm -1 indicates C-H deformation of methyl and methoxyl groups. Nevertheless, aromatic ring-stretching peaks at 1422 cm 1 occur in all spectra of humic preparations under examination. A weak absorption appears at 1340-1330 cm-1, which may indicate phenolic OH groups. In the 1266-1225 cm -1 region, double absorption bands appear, both peaks of which can be attributed to the C-O stretching vibrations of COOH and/or of C-N groups in amide III. Humic substances from the inoculated fresh plant material all show a strong C-O stretching absorption of carbohydrates at 1126 and 1034 cm 1, and similar features can be observed in spectra of the dead plant material. Silicate impurities do not seem to contribute to these absorptions, due to the low ash contents of all humic preparations examined. C-H bending absorptions of aromatics appear at 835 cm-1 in all spectra of the S. alternifloraderived humic substances. DISCUSSION Because of extensive biochemical and microbial transformations during early decomposition, humic compounds represent that part of organic matter from which all substances considered to be readily available sources of energy have been exhausted (Rashid, 1985). Thus, if the organic substances isolated by Alberts et al. (1988) and Filip et al. (1988) from living and dead S. alterniflora are considered to be humic compounds, they must also be able to persist during the decomposition of fresh plant material and dead plant debris in sea water. The results of this study show this to be true, both for the plant materials kept for 10 months under sterile conditions and those incubated after inoculation
282 with different microorganisms. Whereas some amounts of humic-like substances have been shown to be released into sea water (Filip and Alberts, 1988), appreciable quantities remain bound in the plant tissues. Epiphytic microorganisms and microorganisms indigenous to mud enhanced the yield of humic substances up to 168% from the fresh plant material and up to 100% from the dead debris. From the carbon content of S. alterniflora (fresh 40.7%; dead, 52.0%; Filip and Alberts, 1988) and from the data in Tables 1 and 2, one can calculate that a maximum of 1.48% of the C of the incubated fresh plant material and 1.20% of the C of the incubated dead plant material was present as humic substances. Since the humic substances content of living S. alterniflora was calculated to be 0.64% of the total plant carbon (Filip et al., 1988), one has to assume an additional humification of the plant biomass by the mixed microbial populations in sea water during the incubation period. For P. typharum growing on the fresh plant material submerged in sea water, 0.82% of the total plant carbon was found in the humic substances. The data for L. obiones were similar, indicating a lower humification activity of these fungi in the submerged plant material. In comparison with the elemental composition of humic substances extracted from fresh samples of living S. alterniflora, which was reported to be 46% C and 5.8% N by Filip et al. (1988), humic substances examined in this study exhibited a higher C content (56.11-58.49%) and a lower N content (2.16-3.05%). This may be due to microbial utilization of simple structures of the plant humic substance during the long-term incubation. The remaining humic compounds may then be more resistant to further decomposition. These differences are not strongly expressed with the humic substances from the dead plant debris. Generally, the elemental data of all humic substances recovered in this experiment correspond well with those reported by others for aquatic or sedimentary humic acids (Ishiwatary, 1975; Stevenson, 1982; Rashid, 1985; Steelink, 1985). The atomic ratio of hydrogen to carbon is related to the percentage saturation of the carbon atoms, and is therefore important for aquatic humic substances. According to Thurman (1985), an H/C ratio of 2:1 indicates that the carbon atoms are aliphatic in character, whereas a ratio of 1:1 indicates aromatic structures. Since the H/C ratios of all the preparations examined in this study are in the range 1.13-1.41, one can assume the presence of aromatic structures. Ertel and Hedges (1985) listed similar values for humic substances obtained from fresh and degraded plant tissues. The O/C ratios give values between 0.40 and 0.57, and the values of N/C ratios are between 0.03 and 0.05, i.e. in a range typical for soil and aquatic humic acids, according to Steelink (1985). The extinction curves recorded with visible light mainly exhibit features which are typical for humic acids (Figs 1 and 2). The small maxima (or shoulders) at 670nm which appear in some preparations (Fig. 1) can be attributed to plant porphyrines, but they are also typical of marine humic acid,
283 according to Ertel and Hedges (1983). The curves in Fig. 3 demonstrate that some humic preparations from this experiment exhibit an extinction curve which is almost identical to that of the mud humic acid, and that the 670 nm extinction of the mud humic acid may originate from humic compounds related to the freshly collected S. alterniflora. The E4]E6 ratios given in Table 3 correspond well with the values found for sedimentary humic acids by Kalinowski and Blondeau (1988). The similarity between the mud humic acid and humic substances extracted from either fresh or dead S. alterniflora decomposing in sea water containing the mixed population of salt marsh indigenous microorganisms can be seen clearly also from Fig. 4. There are common spectral features, shown as absorption maxima or shoulders at 275-280 and 320 nm. Filip et al. (1976) found similar absorption maxima for humic-like fungal melanins, whereas Goldschmid (1971) reported absorbance in the same regions for phenolic structures of grass lignin. FTIR spectra of the humic substances from S. alterniflora show all the major features typical for humic acids or for model polymers isolated from either terrestrial or marine environments and which were reported by Filip et al. (1974), Stevenson (1982), MacCarthy and Rice (1985), and Thurman (1985). However, the interpretation of amide bands at 1655, 1515 and 1266-1225 cm -1 should be made with care because of the low N contents of the humic preparations tested. In these particular positions, C = O stretching and C -- C aromatic ring stretching must also be considered. The need for caution in interpreting these bands is further emphasized by the fact that it may be calculated for marsh sediment fulvic and hnmic acids that 35 and 20% of the nitrogen, respectively, is in the form of amino acids (Alberts and Filip, 1988). Thus, of the already low nitrogen content of these humic substances, a significant portion may comprise amino acid nitrogen, further reducing the potential amount of amide-bound nitrogen in the compounds. Salt marshes have long been recognized as being among the most productive ecosystems in the world (Odum, 1959). According to McLusky (1981), who referred to several individual authors, the production of the above-ground material of S. alterniflora may be up to 3300 g dry weight per square metre per year. This copious biomass is regularly utilized by numerous species of bacteria and fungi under both aerobic and anaerobic conditions (Gessner and Goos, 1973; Haines and Hanson, 1979). The degradation processes naturally result in a slower or faster mineralization of the plant biomass and enhancement of microbial numbers (Newell et al., 1985; Chrzanowski and Spurrier, 1987). However, our results show that there is also organic matter in the tissue of S. alterniflora which apparently resists degradation. This organic matter remains stable during long-term decomposition of plant fragments in sea water, and shows characteristics which are typical for sedimentary humic acids. The amounts of these humic substances increased substantially in the experimental variants inoculated with mixed populations of microorganisms indigenous to salt marshes.
284 T h e s e findings a g r e e well w i t h the thesis f o r m u l a t e d r e c e n t l y by H a t c h e r a n d S p i k e r (1988), w h i c h claims t h a t r e s i s t a n t f r a c t i o n s of fresh p l a n t m a t e r i a l are c o m p a r a b l e to h u m i c s u b s t a n c e s w i t h i n t h e c o n t e x t of t h e d e g r a d a t i v e s c h e m e for h u m i f i c a t i o n , b e c a u s e t h e y m a y b e c o m e e n r i c h e d d u r i n g d e g r a d a tion. F u r t h e r m o r e , o u r r e s u l t s b r i n g e x p e r i m e n t a l s u p p o r t to t h e a s s u m p t i o n m a d e by C r a f t et al. (1988), u s i n g s t a b l e i s o t o p e s of c a r b o n and n i t r o g e n . A c c o r d i n g to t h e isotopic c o m p o s i t i o n of m a r s h soils, t h e a u t h o r s deduced t h a t t h e m a r s h e m e r g e n t v e g e t a t i o n a p p e a r s to be t h e p r i n c i p a l s o u r c e of o r g a n i c m a t t e r in soils of t h e s a l t m a r s h e s . F r o m o u r results, a n d w i t h r e s p e c t to the a n n u a l yield of S. alterniflora d r y m a t t e r in a s a l t m a r s h as g i v e n by M c L u s k y (1981), one c a n c a l c u l a t e a p o t e n t i a l a n n u a l i n p u t of a b o u t 3 3 0 k g of fresh S p a r t i n a - r e l a t e d h u m i c s u b s t a n c e s p e r h e c t a r e . T h e r e is no d o u b t t h a t s u c h a n i n p u t m a y s u b s t a n t i a l l y c o n t r i b u t e to t h e l o n g - t e r m p h y s i c o - c h e m i c a l a n d b i o l o g i c a l s t a b i l i t i e s of e s t u a r i n e s a l t m a r s h e s . ACKNOWLEDGEMENTS T h e a u t h o r s t h a n k Dr H.P. R e i s e n a u e r of t h e U n i v e r s i t y of G i e s s e n for the F T I R m e a s u r e m e n t s a n d Dr R. S m e d - H i l d m a n n ( L a n g e n ) for s u p e r v i s i n g the i s o l a t i o n of h u m i c s u b s t a n c e s . T h e y also a c k n o w l e d g e t h e skilled t e c h n i c a l a s s i s t a n c e of Miss M. P r i c e (Sapelo I s l a n d ) a n d Miss A. H e e r (Langen). T h i s r e s e a r c h w a s funded, in p a r t , by the S a p e l o I s l a n d R e s e a r c h F o u n d a t i o n a n d t h e D e u t s c h e F o r s c h u n g s g e m e i n s c h a f t , Bonn. This p a p e r is a j o i n t c o n t r i b u tion of t h e U G A M a r i n e I n s t i t u t e (No. 625) a n d the I n s t i t u t fiir Wasser-, Bodenu n d L u f t h y g i e n e des B u n d e s g e s u n d h e i t s a m t e s . REFERENCES Alberts, J.J. and Z. Filip, 1988. Sources and characteristics of fulvic and humic acids from a salt marsh estuary. Sci. Total Environ., in press. Alberts, J.J., Z. Filip, M.T. Price, D.J. Williams and M.C. Williams, 1988. Elemental composition, stable carbon isotope ratios and spectrophotometric properties of humic substances occurring in a salt marsh estuary. Org. Geochem., 12: 455-467. Chen, Y., N. Senesi and M. Schnitzer, 1977. Information provided on humic substances by EJE 8 ratios. Soil Sci. Soc. Am., 41: 352-358. Chrzanowski, T.H. and J.D. Spurrier, 1987. Exchange of microbial biomass between a Spartina alterniflora marsh and the adjacent tidal creek. Estuaries, 10: 118-125. Craft, C.B., S.W. Broome, E.D. Seneca and W.J. Showers, 1988. Estimating sources of soil organic matter in natural and transplanted estuarine marshes using stable isotopes of carbon and nitrogen. Estuarine Coastal Shelf Sci., 26: 633-641. Ertel, J.R. and J.I. Hedges, 1983. Bulk chemical and spectroscopic properties of marine and terrestrial humic acids, melanoidins and catechol-based synthetic polymers. In: R.F. Christman and E.T. Gjessing (Eds), Aquatic and Terrestrial Humic Materials. Ann Arbor Science, Ann Arbor, Michigan, pp. 143-163. Ertel, J.R. and J.I. Hedges, 1985. Sources of sedimentary humic substances: vascular plant debris. Geochim. Cosmochim. Acta, 48: 2065-2074. Fallon, R.D. and J.K. Pfaender, 1976. Carbon metabolism in model microbial system from a temperate salt marsh. Appl. Environ. Microbiol., 31: 959-968.
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