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Fatty acid composition of organic detritus from Spartina alterniflora
E&urine
and Coastal Marine Science (1973) I,
177-190
Fatty Acid Composition of Organic Detritus from Spartina alterniflora
David M. Schultz and James G. Quinn Graduate School of Oceanography, University Kingston, Rhode Island 02881, U.S.A.
of Rhode Island
Received 1 March 1973
The fatty acid composition and concentration of the marsh grass Spartina aZternijZora were determined monthly (January to December, 1971) on samples of stems and leaves collected from Bissel Cove, Narragansett Bay, Rhode Island. Seasonal differences were found in both composition and concentration. The fatty acids present in suspended organic matter collected at the mouth of Bissel Cove during ebb and flood tide for the same period have also been analyzed. Laboratory model studies involving the formation of suspended organic matter by the decomposition of S. altern$ora have been investigated under aerobic and anaerobic conditions, using both organic solvent-extracted and non-extracted grass. Field model studies were also conducted on marsh grass decomposing in Bissel Cove, using both extracted and non-extracted grass. The fatty acid composition and concentration of suspended organic matter formed in the various model studies were similar to those found in the natural material and differed markedly from that of S. altemi’ora. The total fatty acid concentration and the branched chain IS-carbon acids of both natural and model suspended matter gave higher values than those found in S. alterniflbra. These branched chain acids appear to be the result of microbial activity and their high levels in the suspended organic matter, together with the fatty acid concentrations of the model studies using extracted marsh grass, indicate that micro-organisms are very important in the formation of the natural organic detritus.
Introduction Salt marshes of the Atlantic and Gulf Coasts of North America have been thoroughly studied. The extensive marshes in Georgia have received a great amount of attention and, in fact, a review of many studies in this state has served as the summary statement of ecological energy flow in tidal marshes dominated by the cord grass, Spurtim aZternijIoru (Teal, 1962). Similar treatment has been afforded typical mangrove marshes in Florida (Heald, 1971; Odum, 1970~). However, the New England salt marshes are much smaller than those in Georgia and show to a great extent the effects of drastic seasonal changes (Jeffries, 1972; Nixon & Oviatt, 1973). In general, these authors indicate the relatively small amount of utilization of the grasses by grazing and their importance in the detritus food chains in the New England marshes. In his summary, concerning the production in coastal waters of the world, Mann (1972) states that “over 90% of the marine macrophytes’ production enters the food chain as dissolved or particulate organic matter”. The definition of the term organic detritus, as presented by Darnell (1967u), is given as ‘all types of biogenic material in various stages of microbial decomposition which represent 177
D. M. Schultz &‘J. G. Quinn
potential energy sources for consumers’. This definition is a very broad one and includes a range of detrital particle sizes. However, Odum (1968) has shown that the size utilized by most organisms is rather narrow and in general less than zoo pm in largest dimension. Organic detritus has been shown to be used as a food source by a variety of organisms, including the striped mullet (Odum, 1970b) and deposit feeders (Newell, 1965). This detritus, rich in microbial growth and products, is a better food source for animals than the grass tissue that forms the base of the particulate matter (Odum & de la Cruz, 1967; Darnell, 1967b). S. alte&Jlora plays an important role in this detritus food chain. The fatty acid composition of S. aZterniJora has been reported by several workers (Maurer & Parker, 1967; Jeffries, 1972) and the decomposition of this marsh grass, under both laboratory and field conditions, has been studied (Ustach, 1969; Burkholder & Bornside, 1957; Odum & de la Cruz, 1967). In addition, Jeffries (1972) found that, on the basis of fatty acid analyses, the marsh fishes Fund&s majalis and F. heteroclitus must consume a 5 : I mixture of detritus to the shrimp Palaertwnetes pugio, to account for the fatty acid composition of their gut contents. All of these workers have stated that the probable major source of the organic detritus is the breakdown products and subsequent microbial decomposition of S. alterniJEorastems and leaves. The operational term that will be used in this paper to describe the organic detritus (suspended organic matter) is retained organic matter (Schultz & Quinn, 1972). This material is obtained by filtering a sample of water and includes all organic matter retained in or on the filter, such as living and dead plankton, organic particles with their associated organisms, organic matter/mineral and microbial/mineral combinations, organic colloids and macromolecules and organic matter which has been sorbed or trapped in the process of filtering by the above material or the filter itself. The term retained organic matter is preferred over particulate organic matter for two reasons: (i) it includes sorbed or trapped organic matter and (ii) it does not imply a separation of particle sizes. Depending on the type and initial pore size of the filter after a certain volume of solution has passed through the filter the pore size is reduced by trapped particles, etc. and these particles as well as the filter and material on the filter surface can sorb or trap organic matter. The purpose of this study was to compare the qualitative and quantitative distribution of the total fatty acids in suspended organic matter found naturally in the waters of Bissel Cove, Narragansett Bay, Rhode Island, with suspended organic matter produced by laboratory model (aerobic and anaerobic) and field model studies of the decomposition of S. alterniflora. These comparisons would provide information on the role of S. alternifora in the formation of organic detritus in estuaries. Fatty acids were used as the organic tracer in these studies because they are relatively stable, sensitive and accurate methods of analysis are available and the fatty acid composition of terrestrial detritus is very different than that found in tissues of marine organisms. Jeffries (1972) used the fatty acid composition of SUSpended organic matter and marine organisms from Bissel Cove to show that the fatty acids could be used as tracers in establishing estuarine feeding relationships. The fatty acid composition of suspended organic matter collected monthly during both ebb and flood tides at the mouth of Bissel Cove (this area has been described by Nixon & Oviatt, 1973) was measured over a one-year period (January to December, 1971). The fatty acid composition of S. aZterniJlora was determined on samples collected monthly from the shore of the cove during the same year period. Retained organic matter produced in laboratory and field model studies on the decomposition of the marsh grass were also analyzed for fatty acids.
Organic detritus from Spartina alterniflora
Materials
179
and methods
Natural retained organic matter was obtained by filtering water samples containing suspended organic matter, collected from approximately the top zo cm of water surface. These samples were collected monthly in 4-liter glass containers at both ebb and flood tide from the mouth of Bissel Cove entering Narragansett Bay. Approximately 3 liters of each sample was filtered through a preweighed, ignited (4 h at 450 “C) Gelman type A glass fiber filter (initial pore size approximately 0.3 pm) within approximately 2 h of collection. After filtering, the filter was rinsed with distilled water (IS ml) to remove salts and dried in a vacuum desiccator overnight at room temperature. It was then weighed to determine the dry weight of the retained matter on the filter. The filter was placed in a zs-ml culture tube equipped with a teflon-lined screw cap and analyzed for total fatty acids (free and esterified) according to the saponification and methylation procedure of Schultz & Quinn (1972). A second saponification of a retained matter sample showed that all the fatty acids present had been extracted by the first treatment. A blank, consisting of solvents and a filter, contained 14 : o, 15 : o, 16 : o, 16 : I, 18 : o and 18 : I fatty acids (all fatty acids are given by n-carbon chain length : number of double bonds). The sample acids were at least 4 times higher than the blank, averaging 8 times higher, and results in Tables 2 and 3 have been corrected for this blank. S. alternijova samples, combined stems and leaves, were collected monthly from Bissel Cove, rinsed with distilled water, cut into about 2-3 cm pieces and stored at -20 “C until analysis. The S. alterniflora samples (approximately 3 g dry wt, 5-22 g wet wt) were placed in 25o-ml glass round bottom flasks. These flasks and all other glassware used in this study were pre-rinsed with 0.1 N-HCl and IO ml each of methanol, chloroform and methanol to remove fatty acid contaminants. All solvents were ACS reagent grade and were freshly distilled through a 3o-cm glass distillation column (Widmer) prior to use. To the flask containing the marsh grass was added 67 ml of 0.1 N-KOH in methanol and 33 ml of benzene. Internal standard fatty acid (400 l.tg 17 : o) was added and the sample refluxed for 60 minutes. The solution was filtered through a Whatman No. I filter paper into a r-liter separatory funnel and the flask and filter rinsed thoroughly with IOO ml of 0.3 NHCl (extracted previously with CHCI, to remove fatty acid contaminants), 30 ml of methanol and 30 ml of petroleum ether. The filtrate and rinses were combined in the separatory funnel. The benzene/petroleum ether phase was drawn off into a 25o-ml round bottom flask and the acidic, aqueous methanol solution extracted three times with 3o-ml portions of petroleum ether which were added to the initial organic phase. The combined benzene/ petroleum ether extracts were evaporated on a flash evaporator. The residue was then transferred to a rz-ml centrifuge tube equipped with a teflon-lined screw cap using 3 ml of benzene and 3 ml of methanol. Methylation (2 ml of 147; BF, in methanol), isolation and purification via thin-layer chromatography (t.1.c.) was carried out by the same method as for the fatty acids in the retained organic matter (Schultz & Quinn, 1972). A blank, consisting of the same amount of solvents used, was carried through the procedure and showed only trace quantities of fatty acids. In all five model studies, the samples of S. alterniflora were taken after the grass had died. These samples were rinsed, cut and stored as previously described for the monthly samples. The first study involved an aerobic laboratory model system, in which 500 g (dry weight) of S. alternzjloru were placed in approximately IO liters of Bissel Cove water previously filtered through a Whatman No. 54 filter. The filtration allowed most of the microbes to pass through with the water, since the initial pore size of the filter is approximately 4.5 l.trn
180
D. M. Schultz &3J. G. Quinn
(Quinn & Meyers, 1971). The system was aerated continuously and stirred daily. Salinity (20x,), dissolved oxygen (6.5 ml/l) and pH (7.5) were monitored and remained nearly constant for the duration of the experiment. Samples were taken from this system at weeks I, 2, 3, 5, 6, 8, IO, 12 and 17. The samples were first filtered through a 25o-pm stainless steel screen to remove large pieces of grass since most samples of natural suspended organic matter had been found to entirely pass through this screen (large particles were removed from all other samples). The samples were then filtered through an ignited Gelman type A glass fiber filter and treated in an identical manner to the retained organic matter. The second model study was an anaerobic laboratory model system in which samples of 4 g (dry weight) of grass were placed in amber glass bottles, with Whatman No. 54 filtered Bissel Cove water (250 ml). The bottles weresealed immediately and stored out of direct light at 23 “C. Bottles were opened on weeks 3,6, IO and 17 and the samples treated in an identical manner to the aerobic laboratory model system. The third model study was a field model system, in which 22 g (dry wt) of grass were placed in nylon stockings (approximately 250 pm opening) in 0.5-1.5 m of water, about 15 m from shore at Bissel Cove. Samples were retrieved after I, 2, 3,4, 5, 6, 8, IO, 12, 14, 18 and 23 weeks and treated in an identical manner to the two previous model systems. The fourth model study consisted of an anaerobic laboratory model system composed of about 2 g (dry wt) of S. alterniJora from which the lipids had been previously extracted by repeated treatment with 67 ml of 0.1 N-KOH in methanol and 33 ml of benzene as previously described. This procedure removes most natural lipids (especially free fatty acids and their derivatives) and permits the fatty acids produced by micro-organisms in the decomposition process to be followed without interference from the indigenous S. alternijloru fatty acids. The extracted grass was placed in 25o-ml Whatman No. 54 filtered Bissel Cove water in amber glass bottles. The model system consisted of three separate controls for each sample, (i) extracted grass, (ii) extracted grass and I g of glucose and (iii) I g of glucose. Samples were taken after weeks 4, 8, 12 and 16, and the samples treated as the other model systems. The fifth model study consisted of 5 g (dry wt) extracted grass placed in nylon stockings in Bissel Cove as in the third study and collected after 3, 9 and 13 weeks. The samples were again treated the same as the other model systems. Blanks were carried through the procedure for each model study just as they were for natural retained organic matter samples. The blank fatty acids were the same as found for the previously described blank and the sample acids were at least six times higher than the blank, averaging 30 times higher. The results in Tables 4 to 8 have been corrected for the blank acids. In order to differentiate between free and esterified fatty acids in natural retained organic matter, several of the samples were reacted with 8 ml of 0.1 N-KOH in methanol and 5 ml of benzene for 7 min without BF,/methanol treatment. This procedure converts free fatty acids to their potassium salts and esterified fatty acids to their methyl esters by methanolysis (Gunstone, 1967, p. 130). Pure cultures of three species of fungi which had been isolated from S. alterniflora were obtained from the University of Rhode Island Botany Department (Gessner, 1972); they were Sphaerulina pedicellata, Dendryhiella salina and Mucor sp. The cultures were filtered through ignited Gelman type A glass fiber filters and the fatty acid composition determined by the same method as that employed for the natural retained organic matter. Thin-layer chromatography (t.1.c.) procedures for the isolation and purification of the fatty acid methyl ester samples were as previously described for retained organic matter (Schultz & Quinn, 1972). Gas-liquid chromatography (g.1.c.) was carried out on a HewlettPackard Model 5750 flame ionization gas chromatograph. The instrument was equipped
Organic detritus from Spartina alterniflora
181
with a polar column (I 5% stabilized DEGS on Gas Chrom P, 80/1oo mesh) and a non-polar column (2% Apiezon L on Anakrom Q, go/100 mesh). The columns used were 2.1 m by 32 mm o.d. (2.2 mm i.d.) stainless steel and were operated at 180 “C (polar) and 210 “C (non-polar), with nitrogen carrier gas (flow of 35-50 ml/min at 40-50 lb/in2 g inlet pressure). Fatty acid identifications were made on both columns by comparison of relative retention times of sample methyl ester peaks to those of authentic methyl ester standards and by coinjection of these standards with the sample. Quantitative values agreed before and after hydrogenation using Adam’s catalyst (PtO,). Peak areas of sample methyl ester peaks on the polar column were compared to the area of the internal standard peak (methyl heptadecanoate) to quantitate the results. The areaswere calculated by multiplying peak height by width at half-peak height. Quantitative results with National Heart Institute Fatty Acid Methyl Ester Standard mixture D agreed with the stated composition data with a relative error of less than 5qlo for major components (> 10% of total mixture) and less than 10% for minor components (99% was obtained. The conditions employed in the analysis of the retained organic matter could possibly effect isomerization of some of the unsaturated acids (Ast, 1963). This possibility was checked by analyzing a standard mixture containing 18 : 2 and 18 : 3 fatty acid methyl esters. The composition of the mixture before and after the saponification and methylation procedure was almost identical, indicating that the procedure employed is at least not affecting the 18 : 2 and 18 : 3 unsaturated fatty acids to a measurable extent. In addition, a standard consisting of 18 : 3, 18 : 4, 20 : 4, 20 : 5 and 22 : 6 fatty acid methyl esters was analyzed, before and after the saponification, methylation and t.1.c. procedure. An accuracy and recovery of >99% was found when compared with the initial mixture. Thus, this procedure is suitable for the analyses of marine polyunsaturated fatty acids. Based on 13 analyses (each in triplicate) on the fatty acid weight percentages in S. akerniflora (Table I), it was found that the eleven individual fatty acids detected gave an average coefficient of variation of 18.4%. The total fatty acids (pg/mg) gave an average coefficient of variation of 18*5~/~.The standard deviation (s.D.) for each individual acid can therefore be calculated by multiplying the fatty acid weight percent by the coefficient of variation [e.g. Jan. 16 : o, (19-4O/~) (0.184) = 3.6%; 19.4f3-60/b]. The method employed for the natural retained organic matter (Tables 2 and 3) gave an average coefficient of variation of 18.47; for the ten individual fatty acid weight percentages (based on 17 analyses each in triplicate and 6 analyses each in duplicate) and the total fatty acids (pg/mg) gave an average of 21.1~~. With the model studies (Tables 4 to 8), eleven individual fatty acid weight percentages in the retained organic matter gave an average coefficient of variation of 13*74/o (based on 21 analyses each in triplicate, 3 analyses each in duplicate) and the total fatty acids (pg/mg) gave an average of 12.7%. Therefore, from these 3 average coefficients of variations (the S. alternijlora, natural retained organic matter and the model retained organic matter), an overall average of 16.5% was obtained for the individual fatty acid weight percentages and 17*2O/”for the total fatty acids (based on 60 analyses, 51 each in triplicate, 9 each in duplicate).
IS2
D. M. Schultz
M-7.
G. Quinn
Results The fatty acid composition of Spartim altemi~ora, from Bissel Cove, Narragansett Bay, Rhode Island, determined monthly from January, 1971 to December, 1971, is given in Table I. The fatty acid composition of natural retained organic matter, collected at both ebb and flood tides from Bissel Cove, for the same period is given in Tables 2 and 3, respectively. The three laboratory model studies are based on an aerobic system with S. &ernijoru, an anaerobic system with S. ulternifloru and an anaerobic system with extracted S. ulterni$oru. The fatty acid composition for the retained organic matter produced in these model studies are reported in: Table 4 (aerobic, S. uZterniJoru), Table 5 (anaerobic, S. uZterniJoru) and Table 6 (anaerobic, extracted S. uhrnijloru). The results of the field model studies are presented in Table 7 (5’. uZternifZoru) and Table 8 (extracted S. ulterniJoru). The fatty acid composition of the three fungi species isolated from S. ulterniforu are presented in Table 9. TABLE I. Bissel Cove, Narragansett Bay, Rhode Island. Fatty acid weight percentages of Spartina aZterniflo~u (stems and leaves)
Fatty acid“
Jan.
Feb.
Mar.
April
May
14 :o ‘Sb 16 : o
2’9 0.5 19’4 5.4 0.8 5.1 28.3 24’3 7.9
4.8 1.4 29.1 5.4 0’7 8.9 22.7 12’9 6.7 2.5 5.0
3.4 2.4 19’4 6.0 0.8 7.1
2.7 0’9 18.9 5’0 0.7 6.4 22.9 25’45 8. 4.6 3.9
0.9 0.7
16
: I
16 18 18 18 18
: : : : :
20
:o
22
:o
2 0 I 23
2.9 2.7
21’0 22'0 9.0
3.2 5.6
25’1
n.d.’ 0.3 1.8 6.3 ;I’; .
1971 June July 0.8 1.3 20.5
n.d. 0.6 2.1 4.5 y2.z .
1.5
1.3
n.d.
n.d.
Aug.
Sept.
Oct.
Nov.
Dec.
1'2
0.9
1.6 24’4 n.d. 0.7 2.1 4’0 2::;
1.1
1.6 0.4
1.9 n.d. 24.1 2.2
2.2 2.1 21.4 3.0 0.7 3’4 11.7
2.4 0’9
1.6 n.d.
25.6 1.7 tr 2.6 3’7
22'1
;;.z 2.2
Ti.82 1.6
1.0
2'0
1.2 0.4 2.6 6.0
1’1
3.1 7.8
22'0
4’2 0.6 4.8 22.4
25’5 J;‘; I’5 0.8
j;‘; '
11.8
2.5
2'0
1’0
3.6
Total pg/mg;” (SD.)
’ All fatty acids given by normal carbon chain length: number of double bonds. b Consists of undifferentiated species, including both normal and branched (iso and anteiso) r5-carbon acids and a small amount of 14 : I fatty acid. c n.d. =None detected. d pg fatty acid/mg dry weight Spurtim ulterniflo~u (standard deviation).
The monthly survey of S. ulternifloru stems and leaves (Table I) indicates two major trends. (i) The live (May-October) marsh grass is higher in fatty acid concentration (approximately 3-8 pg fatty acid/mg dry wt grass) than the dead grass (December-April, I-Z pg fatty acid/ mg dry wt grass). (ii) Although the fatty acid weight percentage composition generally agrees with published values (Maurer & Parker, 1967; Jeffries, 1972), there are rather drastic seasonal differences in the fatty acid composition between the live and dead S. ulttmi’ru leaves and stems. Among the major compositional differences are the decrease in 18 : 3 fatty acid from an average of 43.4% in the live samples to an average of 8.8% in the dead
Organic detritus from
Spartina
alternitlora
I83
grass. There is also a change in the amount of 18 : I acid percent, from an average of 5.4% (live) to 23’5% (dead). Similar trends have been reported by Jeffries (1972) for samples taken in February and June, 1970. TABLE z. Bissel Cove, Narragansett Bay, Rhode Island. Fatty acid weight percentages of natural retained organic matter-ebb tide
1971” June July
Aug.
Sept.
16.3 77
29.1
19.9
16.9
4.0
4.5
2.8 29'1 30'2 3'3
5.8 47'7 6.9 I'3
1.6
IO’I
4'4
4'2
3'2 36.1 17.0 0.8 2'7 4'3
5.3 3.6
tr.c n.d.
2’9 n.d.
3.9 35'5 22'1 1.9 2'0 8.2 2'0
6.7 2.6 43'0
Fatty acid
Jan.
Feb.
Mar.
May
I4 :o IS* 15 : 0 16 : o
17’9
8.8 3.6 2.5 39'9 33'9 I'9 2.4 5'5 1.8
15.1 47
16 : I 16 :2 18
: 0
18 : I 18
: 2
18
:
3
Total l.tg/mg;” (SD.) mg/le (S.D.)
2.6 2.4 38.6 19.1 3'3 3.2 2'1 8.8 2.1
n.d.
n.d.
10’0
Oct.
Nov.
Dec.
8.1
9.9 4.0
10.4 3.1
12.6
1.9 3.2 49'2 8.7
2.6 36.8
n.d.
3.5 3'1 5'3
2.5 9'4
15.8 0.4
5'3
4.3
3.7
12.7
4.6 16.6 2.6 6.7
2.9 34'9 11.2
2.4 9.2 14'9 6.2 4'9
1.8 I'5 34'9 14.5 2'1 25 13.2 7'0
10’0
(::;)
(t.:)
(2)
(:.i)
(E)
(:*I)
(:I;)
(2)
(2)
(;‘::)
(T::)
(2;)
(I”.;,
(g)
;“,p,,
(8::)
;::;,
;:.:,
(X.i)
(2)
(2)
(2)
’ April sample lost in analytical procedure. b Consists of undifferentiated species, including branched anteiso and iso, and a small amount of I4 : I fatty acid. c tr. =trace. d fig fatty acid/mg dry wt retained matter. e mg dry wt retained matter/liter Bissel Cove water.
I 5-carbon
fatty
acids,
TABLE 3. Bissel Cove, Narragansett Bay, Rhode Island. Fatty acid weight percentages of natural retained organic matter-flood tide
1971"
Fatty acid
Jan.
Feb.
Mar.
May
June
July
Aug.
Sept.
Oct.
Nov.
Dec.
14 :o 19 15 :o
13'9 2.1 1'4
14'5 3.8 2.3
14'9
15.8
26.7 2.6
22.8 3.2
12'1 6.7
4.6 2.9
12.9 3.8
8.8
11.7
1.6
2.0
2.8
6.9 4.5
16 16 16 18 18
36.1
32'5 24'5
32.1
40'5
12.7 1.7
6.2
14.8
4'7
3'4
1’1
2’1
: : : : :
o I 2 o I
18 :2
18 : 3 Total pg/mgC (S.D.) w/l* (S.D.)
1.8
1.8
1.8
3.0
36.1 2.3
2.4 4'2
4'7 7'3
2'2 3.8
9'2 7.4
25'4 27' I I'4 I'2 7.6
23
4.2 3.2
3.1 0.9
I'3 tr.
2.8 3'4
6.8 tr.
1'2 1.7
21.8
13.7
7.8
',"';)
(1.5)
(;.;I
(;:;)
(1.1)
2.6 (0.4)
(0.6)
(z.i)
(1.1)
(1.3)
(i.:)
;T:t)
30.8
6.2
5.5
12.2
15.8 2'2 6.1 15'2
6.3 8.9
i:i 14.1
2.1
4.2 30'3 13.5
3'2 15'4 1.6
6.7 9'4 6.8
7.6
(0.0)
(3.2)
(0.1)
(07)
(2.8)
;z.z)
(:I:)
;y:z)
(t.2)
(i:i)
’ April sample lost in analytical procedure. b Consists of undifferentiated species, including branched and iso, and a small amount of 14 : I fatty acid. c pg fatty acid/mg dry wt retained matter. d mg dry wt retained matter/liter of Bissel Cove water.
2'2 3'0
15-carbon acids, anteiso
D. M. Schultz &f J. G. Quinn
184
The May sample was the first of the new growth and the frost did not kill the grass until the end of October. However, the November sample, although taken after the first frost, still reflected the fatty acid pattern of the live grass more than that of the dead grass. The fatty acid concentration (4.2 pg/mg) was still high and the weight percent of the 18 : 3 acid has only dropped to 33.2% with that of the 18 : I acid (11.7%) being intermediate between the live and dead grass. TABLE 4. Fatty acid weight percentages of retained uZternifIora aerobic laboratory model system
organic matter from Spartina
Weeks Fatty acid 14 15 I5 16 16 16
:o
18
:o
I
2
2’9 0.5
3.1 8.1
2.1 8.2
tr.
:o :o : I
19’4 5’4
: 2
0.8
2.8
I'2
17’4 14’0
2’7
23’5 12.4 0.6 4.0 34.6 7.1 I.4 2.4
1'2
9’9
5’1 28.3 24’3
I8 : I I8 :2 18 : 3’ 22
0
IO.8
:o
Total pg/mgb
1.5 2’4 43’0 2.9 2'1
2.
3
5
6
8
8.0 8.8 3’4 23.2 14’5
2.9 10.9 I’7 22.5 19’5
2.6 8.4 I’3
g:;:
21'1
22.4
I.5
;:;
::i
3’8
22’9 2.8 3.1 3’5
26.4 3.6 2.2 3’2
29.6 2.7 I.9 4’9
5’9
5’1
5’2
IO 5.2
12 6.9
8.1 I’5 25.1 18.3 I.7 6.2 20.5 2.2
1’1 23.0 18.7
;:;
I’3 20.9 18.7 2'1
I7 7’5 8.4 2'2
18.1 20’2
;:;
5’9 25.8 I’9 2.8 5’7
23’4 3’0
. :.:
25.1 4.2 4’7 7’0
3’3
2’4
32
3.2
;:;
a Consists of both I8 : 3 and 20 : o fatty acids. b pg fatty acid/mg dry wt retained matter. TABLE 5. Fatty acid weight percentages of retained organic model system
matter
from
Spartim
aZternifIoru anaerobic laboratory
Weeks Fatty acid 14:o I5 ‘5 :o 16 : o 16 : I 16 : 2 18:o 18 : I
0
3
6
2.6
4’3 10'0
4’0 13’9 3’1 23-3 20.9 3’0 5’9 22.6
I’5 1.6 19.5 5’3 I '0
6.9
18:2
32'4 I9.I
18 : 3
IO'2
20
:o
Total pg/mg’ (S.D.)
tr.
(2)
2.6 21'1
29.0 2’7 2’5 24'7 1'3 0.8
;:; (0.3)
I'3 1'0
IO
4’4
5’4
14.8
IO.8
4’2
2’3 47‘4 7‘4 5’4 10.4 8.7
21'0
21.5 7’1 2.8 17.8 2'1 I.3
I’I . (&)
17
1'1
;:::,
n.d. n.d. z-3 (2)
LIpg fatty acid/mg dry weight retained matter.
The dead grass remains standing through the winter until the spring thaw. It is then sheared off and transported from the cove into the bay in fairly large rafts by tidal action (Nixon & Oviatt, 1973).However, the grass in these rafts is largely intact and one would not expect a very abrupt change in the suspended matter content of the water. In addition, large
Organic detritus from Spartina
alterniflora
185
rafts are stranded on the shores of the cove and are slowly removed by the tidal action, thereby providing a source of suspended organic matter over an extended period of time. In order to determine what percentage of the fatty acids in the retained organic matter is contributed by dissolved fatty acids retained by the filter, an experiment was conducted using two filters, one on top of the other. After Bissel Cove water was passed through the filters, both filters and the filtrate were analysed for fatty acids. Based on the amount of fatty acids retained by the second filter in this study, approximately zoo,&of the values reported in this work result from retention of dissolved fatty acids by the Gelman A filter. TABLE 6. Fatty acid weight percentages of retained organic matter model system
from extracted’
Spartim alternifora anaerobic laboratory Weeks Fatty acid 14 :o
2’4
15
I5 16 16 16 18 18 18 18
:3
20
:o
8
12
16
“5
7’7 8.0 4.2 32.2 IO.4
2'2
4
:o :o : I :2 :o : I : 2
Total ug/mgb
11'5
II'2
1’2 28.5 8.7 6.2 7’0 15’7 18.9 n.d. n.d. 6.0
1.6 19’5 9.5 2’7 4.8 29.2 ZO'I
nd. n.d. 4’0
;:; 16.9 5’3 n.d. 5.1 10.9
6.0 0.9 30’4 13.1 2’7 7’4 12.5 25.0 nd. n.d. 22.6
‘Extracted by repeated treatment with organic material. b pg fatty acid/mg dry wt retained matter.
solvents to remove
TABLE 7. Fatty acid weight percentages of retained organic matter ~Zternifloru field model system (9 June to 17 November 1971)
Fatty acid :o
o
1
z
3
4
2.9 1’4 1’0 19’9 7’2 0.5 7’3 15.6
4.1 4’7 3’3 26.9
4.6 7’4 4’0 27.6 15’5
5.4 15.8 4.2 22.6 14’3 2.8 5’9 16.7 2’9 1.2 2.6 6.0 ’ (i.T)
14 ‘5 I.5 16 16 16 18 18 18 18
:0 :o : 1 :2 :o : 1 :2 :3
20 22
:o :o
7.2 7’7
Total pg/mg’ (SD.)
(0.3)
20'5 8.2
2.2
3.4 3-3 3’4
I% 3’0
5.1 7’5 5’5 25.0 15’4 2.8 5’0 22.6 3’0 I.9 1.8 4’3
6.7 (0.8)
. (L.:)
6.2 (0.5)
11’0 1.0
1.2
6.6 28.2
5’3 26.7
4'1
. 2.1
5
Weeks 6
z::
2.2
11.4 4’9 n. d . 3.6 n.d.
12.5 n.d. n.d. n.d. n.d.
5.0 4.1 13’3 26.2 18.0 6.2 2.8 15.6 1.9 “3 1.2 4.4
6.0 (0.9)
. (‘0.:)
6.2 (0.9)
5.7 22'2
6.1 25.9 IO.9
7.8 6.6 16.6 30.1 ‘4’7 9.5
8
u pg fatty acid/mg dry weight retained matter.
natural
lipid
from Spartim
14
18
23
5.8 9’4 4.2 26. I 16.5 7.5 3.4 21.8 2.0 1.7 nfc? .
7.2 7’5 5.1 30.8 18.4 6.9
2.9 4.4 tr. 4.4
7.1 10’2 7.6 26.0 16.2 4’9 3.5 16.9 1.7 2.6 n.d. 3.3
2.2
6.5 12.6 3.6 28.8 21.9 4.4 14 16.3 0.6 1’7 n.d. 1’7
8.0 (1.3)
(:I:)
(i.i)
(t.z)
(i.:)
IO
12
5.1 4.5 4.8 44.8 II'0
3.8 3.3 II'2
2.0
17.2 0.9 1.8 n.d.
186
D. M. Schultz &J. G. Quinn
The natural retained organic matter showed little difference in the fatty acid compositions between the ebb (Table 2) and flood tides (Table 3). For example, the 16 : o fatty acid (&S.D.) averaged 38*7-&5+9% in the ebb tide samples and 33+3&5.8% in the flood tide samples, and the total Is-carbon acids (anteiso, iso and normal) were 7-I&2*7% (ebb) W~YSUS 6*2f2*5% (flood). The 14 : o acid average was 15*0&6~1‘J(~(ebb) and 14*4&6*1’7~ (flood) and the 18 : I average was 8*0&-4*9% (ebb) and 9*1&3*7% (flood). However, the total fatty acid concentration (pg FA/mg dry wt retained matter) of the flood tide was significantly higher (at the 95% confidence level) than that of the ebb tide in 7 of the II samples (ebb tide-average 5*0&1-3 pg/mg, flood tide-average 7*9& 1-6 l.tg/mg). In addition, the data in Tables 2 and 3 indicate a general flux of retained matter outward from the marsh into the bay (significant at the 90% confidence level in 8 of the II samples). The values of mg dry weight retained matter/liter of Bissel Cove water are ebb tide-average 8.of1.7 mg/l and flood tide-average 6.0% I-O mg/l. The ebb tide appears to be transporting suspended matter from the cove into the bay which, however, is lower in fatty acid concentration than that already in the bay. This might indicate that the suspended matter transported from the cove to the bay has less microbial growth associated with it and may be recently synthesized detritus produced from the dead marsh grass. TABLE 8. Fatty acid weight percentages of retained organic matter from extracted” Spartim alterniflora field model system (3 August to z November 1972) Weeks Fatty acid 14 :0 I5 15 16 16 16
:o :o : I :2
18 : o 18 : I 18 : 2 18 : 3 20 : 0 Total l.tg/mgb
3
9
13
8.0
5’4
7’0
32.1
13.0 6.7 26.2
24’4 4.9 1.6 15’3
23’5 2.8 I’9 20.7
8.1 9’4 27.0 20’7
n.d. n.d. n.d.
n.d. n.d. n.d.
9’7
3’1
a::
2.7 3’0 17.1 1.6
I’S 2’0 2’2
a Extracted by repeated treatment with organic solvents to remove natural material. b l,tg fatty acids/mg dry wt retained matter.
lipid
The effect of phytoplankton blooms in the bay on the fatty acid composition of retained organic matter appears to be fairly small, since the amount of detrital material from plant decomposition is much larger than the amount of living phytoplankton contained in 2-4 liters of water from the mouth of the cove. Since there were none of the marine polyunsaturated acids (such as 20 : 5 and 22 : 6; Jeffries, 1970) found in the retained organic matter samples, this observation seems valid, although it must be re-emphasized that these polyunsaturated acids would probably be masked by the large amount of terrestrial suspended matter present in the water in Bissel Cove. There were also large amounts of 18 : 3 found in several of the samples of retained organic matter (e.g. September) which can not be explained at this time.
Organic
detritus from
Spartina alterniflora
187
The fatty acid composition of marsh grass (Table I) differs from that of the natural retained organic matter (Tables 2 and 3) in several major areas. The marsh grass generally has more of the unsaturated acids 18 : I, 18 : 2 and 18 : 3 than is found in the retained organic matter. Both the 14 : o and 16 : o acids showed lower trends in the marsh grass than in the retained organic matter. In addition, the unsaturated 16 : I acid and the total Is-carbon acids tend to be much lower in the marsh grass than in the retained organic matter. The relatively long chain saturated acids 20 : o and 22 : o were present in the marsh grass but not detected in the retained organic matter. TABLE
9. Fatty
acid weight
percentages
of fungi
species isolated
from
Spartinn
alternifEora
Fatty Acid I4 : 0 15 *5 16 16 16
:o : o : I : z
18
:o
18 : I 18
:2
18 : 3
Sphaerulina pedicellata
Dendryphiella salina
Mucor species
0.9 3.8
n.d.
n.d. n. d n.d.
n.d. n.d.
20.4
30’2
26.8
4.6 0.9 7’0 27’4 33’0
3’4 n.d.
n.d.
6.4 23.6
40’9
2.0
36.4
n.d.
0.8
1'2
12'0
18.3 n.d.
The procedure for differentiating esterified and free fatty acids was performed on a natural retained organic matter sample collected at ebb tide and provided additional information concerning the nature of the fatty acids present in retained organic matter. Approximately 60% of the fatty acids were present in an esterified form in this sample, compared to >99% present in the esterified form in a sample of dead 5’. aZterniJora. This procedure may be useful in tracing the pathways of organic matter conversion in estuarine environments by differentiating between various sources and processes. Additional studies using this procedure are now in progress. The model studies were designed to compare the natural suspended matter from Bissel Cove with material produced by allowing dead S. altern$ora to decompose under laboratory and field conditions. The studies using extracted marsh grass were performed in an attempt to see if microbial growth on fatty acid-free particles was the most important source of fatty acids in detritus formation. The model studies with the S. aZterni$ora revealed several interesting trends. The aerobic laboratory model system (Table 4) produced material in which the weight percent of the saturated acids 14 : o and 22 : o generally increased over the 17 week period. The 16 : o, 18 : o and 18 : I acids were variable over the time interval, but remained essentially constant. The total Is-carbon acids showed a very marked increase over the time interval, going from o-50/Oinitially to 10*60/Oafter 17 weeks. Among the unsaturated acids, 16 : I increased from 5.4% to 20*2%, 16 : 2 showed a slight increase and both 18 : 2 and 18 : 3 (20 : o) decreased markedly (35.1% total initially to 4.7% total after 17 weeks). Under anaerobic conditions (Table 5), similar trends were observed with the exceptions that the 16 : o acid increased between the tenth and seventeenth week, the 16 : I acid generally increased to IO weeks and decreased at 17 weeks, 16 : 2 increased substantially
188
D. M. Schultz &f J. G. Quinn
1.0 to 7.1~/~) to IO weeks and the 18 : I acid generally decreased over the r7-week period. In addition, the saturated 22 : o acid was not detected in any of these samples. At the end of 17 weeks, the fatty acid composition of the retained organic matter produced by both systems showed values that were considerably different from the initial S. alterniflora. The aerobic system had a high concentration of 16 : I and 18 : I, whereas both these acids were rather low in the anaerobic system and 16 : o and 18 : o acids were higher in the anaerobic than aerobic system. The total concentration of acids (pg/mg) increased from 1.2 to 3.2 pg/mg, with a maximum at I week for the aerobic and increased from 1.5 to 7.6 pg/mg (maximum at IO weeks), for the anaerobic system. These values, 3.2 and 7.6 pg/mg, are similar to most of the values reported in Tables 2 and 3 for natural retained matter, especially those of the ebb tide samples (2-1 to 8.5 pg/mg) which might reflect values of newly synthesized detritus transported from cove to bay as previously described. The trends observed in the field study (Table 7) were similar to those observed for the laboratory studies with most values closer to those of the aerobic laboratory system. Although the 16 : o acid generally increased over the r8-week period, the other saturated acids (18 : o, 20 : o, 22 : o) generally decreased over the time interval. The total concentration of fatty acids increased after I week and then remained essentially constant through 18 weeks. The value (6.3 pg/mg) at I 8 weeks resembled the anaerobic study more than the aerobic study (7.6 and 3.2 pg/mg, respectively) after 17 weeks and also agree with values for ebb tide (Table 2). The trends for the individual fatty acids over the r8-week period generally held after 23 weeks, although the fatty acid concentration decreased during this period. Thus, the available evidence from these model studies indicates the following. (i) Both aerobic and anaerobic processesmay be important in the formation of detritus. (ii) Although the percentage of the 18 : 3 acid decreased under both aerobic and anaerobic conditions, the anaerobic process appears to be important in producing levels of 18 : I and 18 : 2 acids similar to those found for natural retained organic matter. This may be the starting point of suspended matter formation in the marsh, since the sediment/water interface is anaerobic during much of the time in which detrital formation would be occurring. (iii) The total fatty acid concentration @g FA/mg dry wt retained matter) of natural and model retained matter was very similar. (iv) The IS-carbon acids may be the products of microbial activity (Parker et al., 1967) and in a closed system accumulate to a very high level. (v) The concentration of fatty acids generally increases over time, with qualitative changes readily apparent. In order to determine whether the fatty acids being measured were from S. alternijoru itself or bacteria and other micro-organisms living on the particles, a laboratory model system (Table 6) and a field model (Table 8) were investigated using S. ulternifloru from which the fatty acids had been extracted. The intent was to use the extracted grass as a solid support, providing glucose as a carbon source and measure qualitative and quantitative changes in the fatty acids. The initial fatty acid concentration of the grass was about 0.1 pg FA/mg retained matter. After 16 weeks the laboratory model retained matter had a fatty acid concentration of 22.6 pg/mg (Table 6) (aft er correcting for the controls), giving a fatty acid increase of about 200 times over the initial value (22*6/o-1). Since small particles which break loose from the dead grass had essentially the same fatty acid composition and concentration as the intact grass, the increase must be attributed to some type of microbial growth. The fatty acid composition of the laboratory retained organic matter tended to be higher in the unsaturated acids 18 : I and 18 : 2 than the natural retained organic matter but the levels of 16 : 0, 16 : I, 16 : 2, 18 : o and total Is-carbon acids were very similar to the natural
Organic detritus from Spartina
189
alterniflora
retained matter. However, the 14 : o and 18 : 3 acids both tended to be lower in the laboratory retained matter than in the natural samples. In the field study with extracted marsh grass (Table 8), an increase was observed in the fatty acid concentration of the retained matter (about 6 times) after 13 weeks (from 0.3 pg/mg initially to 2.2 pg/mg). The fatty acid composition of the retained organic matter produced in this study showed similar trends to the laboratory study, with the exception that the 14 : o and 18 : 3 fatty acid weight percentages were now also similar to that found in the natural retained organic matter and the 18 : I and 18 : 2 acids were lower than those in the laboratory study. The fatty acid composition of this field study was very similar to that found after 18 weeks in the field study using the non-extracted S. alterniflora (Table 7) and both gave a fatty acid composition and concentration very similar to the natural retained organic matter from ebb tide. Both of these models gave indications that the entire fatty acid concentration and composition of the natural retained organic matter can be accounted for by the activity of associated micro-organisms. The fatty acid composition of the fungi (Table 9) all contain fairly large amounts of 16 : o, 18 : o, 18 : I and 18 : 2, but only Sphaerulina pedicellata contains any IS-carbon acids. This fungus is one of the more abundant fungi isolated from the Bissel Cove salt marsh (Gessner, 1972) and appears to initiate natural fungal colonization of Spartina alternijlora (Gessner et al., 1972). Teal (1962) estimated that in the Georgia marshland, bacterial activities involved in degradation of S. alterniflora accounted for 59% of available plant substrate. However, the ‘bacterial’ activities were not subdivided into bacteria and fungi. The role of fungi in the degradation of plant material has been studied in a fresh water environment and Kaushik & Hynes (1971) state that fungi are more important than bacteria in the initial stages of the degradative process. Some of the acids present in the retained organic matter formed in the model studies could be a result of the presence of these or other fungi species, although only Sph. pedicellata contains any of the Is-carbon acids. It appears that the fungi alone cannot totally account for the fatty acid composition of any of the model studies or the natural retained organic matter. Marine bacteria are high (up to 57%) in the branched (iso and anteiso) Is-carbon acids (Parker et aZ., 1967) and a combination of bacteria, fungi and other micro-organisms probably contributes to the fatty acid composition of the natural retained organic matter. There was fairly good agreement in the fatty acid composition and concentration for retained organic matter produced in laboratory and field model systems and natural retained organic matter. The model systems indicated that the actual formation of the natural suspended organic matter is a very complex process which probably involves both aerobic and anaerobic reactions. In addition, natural organic detritus is a very heterogeneous mixture of material in varying stages of decay and containing various amounts and types of micro-organisms. Acknowledgements This investigation was supported by the National Sea Grant Program (04-3-158-3). We thank Dr H. Perry Jeffries for reviewing the manuscript and Mr Robert V. Gessner for providing the fungi cultures. References Ast., H. J. 1963 Inadvertant
isomerization tical Chemistry 35, x539-15+0.
of polyunsaturated
acids during
ester preparation.
Analy-
190
D. M. Schultz &f J. G. Quinn
Burkholder, P. R. & Bornside G. H. 1957 Decomposition of marsh grass by aerobic marine bacteria. Bulletin of the Torrey Botanical Club 84, 366-383. Darnell, R. M. 1967~ The organic detritus problem. In Estuaries (Lauff, G. H., ed.). American Association for the Advancement of Science, Washington. pp. 374-375. Darnell, R. M. 19676 Organic detritus in relation to the estuarine ecosystem. In Estuaries (Lauff, G. H., ed.) American Association for the Advancement of Science, Washington, pp. 376-382. Gessner, R. 1972 The fungi associated with Spartina alterniflora and other salt marsh plants from southern Rhode Island. Masters thesis, Univ. of Rhode Island. pp. I-IZZ. Gessner, R. V., Goes, R. D. & Sieburth, J. McN. 1972 The fungal microcosm of the internodes of Spartina alterniflora. Marine Biology 16, 269-273. Gunstone, F. D. 1967 An Introduction to the Chemistry and Biochemistry of Fatty Acids and their Glycerides. Chapman and Hall, Ltd, London. pp. 1-209. Heald, E. J. 1971 The production of organic detritus in a south Florida estuary. Sea Grant Technical Bulletin Number 6 Univ. of Miami. pp. I-I IO. Herb, S. F. & Martin, V. G. 1970 How good are analyses of oil by g.1.c. Journal of the American Oil Chemists Society 47, 415-42 I. Horning, E. C., Ahrens, E. H. Jr, Lipsky, S. R., Mattson, F. II., Mead, J. I;., Turner, D. A. & Goldwater, W. H. 1964 Quantitative analysis of fatty acids by gas-liquid chromatography. Journal of Lipid Research 5, 20-27. fatty acids. Limnology Jeffries, H. P. 1970 Seasonal composition of temperate plankton communities: and Oceanography 15,419-426. Jeffries, H. P. 1972 Fatty-acid ecology of a tidal marsh. Limnology and Oceatzogvuphy 17, 433-440. Kaushik, N. K. & Hynes, H. B. N. 1971 The fate of dead leaves that fall into streams. Archiw FZIY Hydrobiologie 68,465-5 I 5. Mann, K. H. 1972 Macrophyte production and detritus food chains in coastal waters. Symposium on ‘Detritus and its ecological role in aquatic ecosystems’ zznd-26th May 1972, Pallanza, p. I-57. Maurer, L. G. & Parker, P. L. 1967 Fatty acids in sea grasses and marsh plants. Contributions in Marine ,%ie?nCe 4, 113-119. Newell, R. 1965 The role of detritus in the nutrition of two marine deposit feeders, the prosobranch Hydrobia ulvae and the bivalve Macoma balthica. Proceedings of the Zoological Society of London 114, (I), 25-45. Nixon, S. W. & Oviatt, C. A. 1973. Ecology of a New England Salt Marsh Ecological Monographs. In press. Odum, E. P. & de la Cruz, A. A. 1967 Particulate organic detritus in a Georgia salt marsh estuarine ecosystem In Estuaries (Lauff, G. H., ed.) American Association for the Advancement of Science, Washington. pp. 383-388. Odum, W. E. 1968 The ecological significance of fine particle selection by the striped mullet Mugil cephalus. Limnology and Oceanography 13, 92-98. Odum, W. E. 1g70a. Pathways of energy flow in a south Florida estuary. Ph.D. dissertation, Univ. of Miami. 126-136. Odum, W. E. Ig7ob Utilization of direct grazing and plant detritus food chains by the striped mullet Mu&l cebhalus. In Marine Food Chains (Steele, J. H., ed.) Univ. of Calif. Press, Calif. pp. 222-240. Parker, ‘I’. L:, Van Baalen, C. & Maurer, L. 1967~Fatty acids in eleven species of blue-green algae: geochemical significance. Science 155, 707-708. Quinn, J. G. & Meyers, P. A. 1971 Retention of dissolved organic acids in sea water by various filters. Limnology and Oceanography 16, I 29-13 I. Schultz, D. M. & Quinn, J. G. 1972 Fatty acids in surface particulate matter from the North Atlantic. Journal of the Fisheries Research Board of Canada 29, 1482-1486. Teal, J. M. 1962 Energy flow in the salt marsh ecosystem of Georgia. Ecology 43, 614-624. Ustach, J. F. 1969 The decomposition of Spartina aZterni$ora. Masters thesis, North Carolina State University. pp. 1-26.
and Coastal Marine Science (1973) I,
177-190
Fatty Acid Composition of Organic Detritus from Spartina alterniflora
David M. Schultz and James G. Quinn Graduate School of Oceanography, University Kingston, Rhode Island 02881, U.S.A.
of Rhode Island
Received 1 March 1973
The fatty acid composition and concentration of the marsh grass Spartina aZternijZora were determined monthly (January to December, 1971) on samples of stems and leaves collected from Bissel Cove, Narragansett Bay, Rhode Island. Seasonal differences were found in both composition and concentration. The fatty acids present in suspended organic matter collected at the mouth of Bissel Cove during ebb and flood tide for the same period have also been analyzed. Laboratory model studies involving the formation of suspended organic matter by the decomposition of S. altern$ora have been investigated under aerobic and anaerobic conditions, using both organic solvent-extracted and non-extracted grass. Field model studies were also conducted on marsh grass decomposing in Bissel Cove, using both extracted and non-extracted grass. The fatty acid composition and concentration of suspended organic matter formed in the various model studies were similar to those found in the natural material and differed markedly from that of S. altemi’ora. The total fatty acid concentration and the branched chain IS-carbon acids of both natural and model suspended matter gave higher values than those found in S. alterniflbra. These branched chain acids appear to be the result of microbial activity and their high levels in the suspended organic matter, together with the fatty acid concentrations of the model studies using extracted marsh grass, indicate that micro-organisms are very important in the formation of the natural organic detritus.
Introduction Salt marshes of the Atlantic and Gulf Coasts of North America have been thoroughly studied. The extensive marshes in Georgia have received a great amount of attention and, in fact, a review of many studies in this state has served as the summary statement of ecological energy flow in tidal marshes dominated by the cord grass, Spurtim aZternijIoru (Teal, 1962). Similar treatment has been afforded typical mangrove marshes in Florida (Heald, 1971; Odum, 1970~). However, the New England salt marshes are much smaller than those in Georgia and show to a great extent the effects of drastic seasonal changes (Jeffries, 1972; Nixon & Oviatt, 1973). In general, these authors indicate the relatively small amount of utilization of the grasses by grazing and their importance in the detritus food chains in the New England marshes. In his summary, concerning the production in coastal waters of the world, Mann (1972) states that “over 90% of the marine macrophytes’ production enters the food chain as dissolved or particulate organic matter”. The definition of the term organic detritus, as presented by Darnell (1967u), is given as ‘all types of biogenic material in various stages of microbial decomposition which represent 177
D. M. Schultz &‘J. G. Quinn
potential energy sources for consumers’. This definition is a very broad one and includes a range of detrital particle sizes. However, Odum (1968) has shown that the size utilized by most organisms is rather narrow and in general less than zoo pm in largest dimension. Organic detritus has been shown to be used as a food source by a variety of organisms, including the striped mullet (Odum, 1970b) and deposit feeders (Newell, 1965). This detritus, rich in microbial growth and products, is a better food source for animals than the grass tissue that forms the base of the particulate matter (Odum & de la Cruz, 1967; Darnell, 1967b). S. alte&Jlora plays an important role in this detritus food chain. The fatty acid composition of S. aZterniJora has been reported by several workers (Maurer & Parker, 1967; Jeffries, 1972) and the decomposition of this marsh grass, under both laboratory and field conditions, has been studied (Ustach, 1969; Burkholder & Bornside, 1957; Odum & de la Cruz, 1967). In addition, Jeffries (1972) found that, on the basis of fatty acid analyses, the marsh fishes Fund&s majalis and F. heteroclitus must consume a 5 : I mixture of detritus to the shrimp Palaertwnetes pugio, to account for the fatty acid composition of their gut contents. All of these workers have stated that the probable major source of the organic detritus is the breakdown products and subsequent microbial decomposition of S. alterniJEorastems and leaves. The operational term that will be used in this paper to describe the organic detritus (suspended organic matter) is retained organic matter (Schultz & Quinn, 1972). This material is obtained by filtering a sample of water and includes all organic matter retained in or on the filter, such as living and dead plankton, organic particles with their associated organisms, organic matter/mineral and microbial/mineral combinations, organic colloids and macromolecules and organic matter which has been sorbed or trapped in the process of filtering by the above material or the filter itself. The term retained organic matter is preferred over particulate organic matter for two reasons: (i) it includes sorbed or trapped organic matter and (ii) it does not imply a separation of particle sizes. Depending on the type and initial pore size of the filter after a certain volume of solution has passed through the filter the pore size is reduced by trapped particles, etc. and these particles as well as the filter and material on the filter surface can sorb or trap organic matter. The purpose of this study was to compare the qualitative and quantitative distribution of the total fatty acids in suspended organic matter found naturally in the waters of Bissel Cove, Narragansett Bay, Rhode Island, with suspended organic matter produced by laboratory model (aerobic and anaerobic) and field model studies of the decomposition of S. alterniflora. These comparisons would provide information on the role of S. alternifora in the formation of organic detritus in estuaries. Fatty acids were used as the organic tracer in these studies because they are relatively stable, sensitive and accurate methods of analysis are available and the fatty acid composition of terrestrial detritus is very different than that found in tissues of marine organisms. Jeffries (1972) used the fatty acid composition of SUSpended organic matter and marine organisms from Bissel Cove to show that the fatty acids could be used as tracers in establishing estuarine feeding relationships. The fatty acid composition of suspended organic matter collected monthly during both ebb and flood tides at the mouth of Bissel Cove (this area has been described by Nixon & Oviatt, 1973) was measured over a one-year period (January to December, 1971). The fatty acid composition of S. aZterniJlora was determined on samples collected monthly from the shore of the cove during the same year period. Retained organic matter produced in laboratory and field model studies on the decomposition of the marsh grass were also analyzed for fatty acids.
Organic detritus from Spartina alterniflora
Materials
179
and methods
Natural retained organic matter was obtained by filtering water samples containing suspended organic matter, collected from approximately the top zo cm of water surface. These samples were collected monthly in 4-liter glass containers at both ebb and flood tide from the mouth of Bissel Cove entering Narragansett Bay. Approximately 3 liters of each sample was filtered through a preweighed, ignited (4 h at 450 “C) Gelman type A glass fiber filter (initial pore size approximately 0.3 pm) within approximately 2 h of collection. After filtering, the filter was rinsed with distilled water (IS ml) to remove salts and dried in a vacuum desiccator overnight at room temperature. It was then weighed to determine the dry weight of the retained matter on the filter. The filter was placed in a zs-ml culture tube equipped with a teflon-lined screw cap and analyzed for total fatty acids (free and esterified) according to the saponification and methylation procedure of Schultz & Quinn (1972). A second saponification of a retained matter sample showed that all the fatty acids present had been extracted by the first treatment. A blank, consisting of solvents and a filter, contained 14 : o, 15 : o, 16 : o, 16 : I, 18 : o and 18 : I fatty acids (all fatty acids are given by n-carbon chain length : number of double bonds). The sample acids were at least 4 times higher than the blank, averaging 8 times higher, and results in Tables 2 and 3 have been corrected for this blank. S. alternijova samples, combined stems and leaves, were collected monthly from Bissel Cove, rinsed with distilled water, cut into about 2-3 cm pieces and stored at -20 “C until analysis. The S. alterniflora samples (approximately 3 g dry wt, 5-22 g wet wt) were placed in 25o-ml glass round bottom flasks. These flasks and all other glassware used in this study were pre-rinsed with 0.1 N-HCl and IO ml each of methanol, chloroform and methanol to remove fatty acid contaminants. All solvents were ACS reagent grade and were freshly distilled through a 3o-cm glass distillation column (Widmer) prior to use. To the flask containing the marsh grass was added 67 ml of 0.1 N-KOH in methanol and 33 ml of benzene. Internal standard fatty acid (400 l.tg 17 : o) was added and the sample refluxed for 60 minutes. The solution was filtered through a Whatman No. I filter paper into a r-liter separatory funnel and the flask and filter rinsed thoroughly with IOO ml of 0.3 NHCl (extracted previously with CHCI, to remove fatty acid contaminants), 30 ml of methanol and 30 ml of petroleum ether. The filtrate and rinses were combined in the separatory funnel. The benzene/petroleum ether phase was drawn off into a 25o-ml round bottom flask and the acidic, aqueous methanol solution extracted three times with 3o-ml portions of petroleum ether which were added to the initial organic phase. The combined benzene/ petroleum ether extracts were evaporated on a flash evaporator. The residue was then transferred to a rz-ml centrifuge tube equipped with a teflon-lined screw cap using 3 ml of benzene and 3 ml of methanol. Methylation (2 ml of 147; BF, in methanol), isolation and purification via thin-layer chromatography (t.1.c.) was carried out by the same method as for the fatty acids in the retained organic matter (Schultz & Quinn, 1972). A blank, consisting of the same amount of solvents used, was carried through the procedure and showed only trace quantities of fatty acids. In all five model studies, the samples of S. alterniflora were taken after the grass had died. These samples were rinsed, cut and stored as previously described for the monthly samples. The first study involved an aerobic laboratory model system, in which 500 g (dry weight) of S. alternzjloru were placed in approximately IO liters of Bissel Cove water previously filtered through a Whatman No. 54 filter. The filtration allowed most of the microbes to pass through with the water, since the initial pore size of the filter is approximately 4.5 l.trn
180
D. M. Schultz &3J. G. Quinn
(Quinn & Meyers, 1971). The system was aerated continuously and stirred daily. Salinity (20x,), dissolved oxygen (6.5 ml/l) and pH (7.5) were monitored and remained nearly constant for the duration of the experiment. Samples were taken from this system at weeks I, 2, 3, 5, 6, 8, IO, 12 and 17. The samples were first filtered through a 25o-pm stainless steel screen to remove large pieces of grass since most samples of natural suspended organic matter had been found to entirely pass through this screen (large particles were removed from all other samples). The samples were then filtered through an ignited Gelman type A glass fiber filter and treated in an identical manner to the retained organic matter. The second model study was an anaerobic laboratory model system in which samples of 4 g (dry weight) of grass were placed in amber glass bottles, with Whatman No. 54 filtered Bissel Cove water (250 ml). The bottles weresealed immediately and stored out of direct light at 23 “C. Bottles were opened on weeks 3,6, IO and 17 and the samples treated in an identical manner to the aerobic laboratory model system. The third model study was a field model system, in which 22 g (dry wt) of grass were placed in nylon stockings (approximately 250 pm opening) in 0.5-1.5 m of water, about 15 m from shore at Bissel Cove. Samples were retrieved after I, 2, 3,4, 5, 6, 8, IO, 12, 14, 18 and 23 weeks and treated in an identical manner to the two previous model systems. The fourth model study consisted of an anaerobic laboratory model system composed of about 2 g (dry wt) of S. alterniJora from which the lipids had been previously extracted by repeated treatment with 67 ml of 0.1 N-KOH in methanol and 33 ml of benzene as previously described. This procedure removes most natural lipids (especially free fatty acids and their derivatives) and permits the fatty acids produced by micro-organisms in the decomposition process to be followed without interference from the indigenous S. alternijloru fatty acids. The extracted grass was placed in 25o-ml Whatman No. 54 filtered Bissel Cove water in amber glass bottles. The model system consisted of three separate controls for each sample, (i) extracted grass, (ii) extracted grass and I g of glucose and (iii) I g of glucose. Samples were taken after weeks 4, 8, 12 and 16, and the samples treated as the other model systems. The fifth model study consisted of 5 g (dry wt) extracted grass placed in nylon stockings in Bissel Cove as in the third study and collected after 3, 9 and 13 weeks. The samples were again treated the same as the other model systems. Blanks were carried through the procedure for each model study just as they were for natural retained organic matter samples. The blank fatty acids were the same as found for the previously described blank and the sample acids were at least six times higher than the blank, averaging 30 times higher. The results in Tables 4 to 8 have been corrected for the blank acids. In order to differentiate between free and esterified fatty acids in natural retained organic matter, several of the samples were reacted with 8 ml of 0.1 N-KOH in methanol and 5 ml of benzene for 7 min without BF,/methanol treatment. This procedure converts free fatty acids to their potassium salts and esterified fatty acids to their methyl esters by methanolysis (Gunstone, 1967, p. 130). Pure cultures of three species of fungi which had been isolated from S. alterniflora were obtained from the University of Rhode Island Botany Department (Gessner, 1972); they were Sphaerulina pedicellata, Dendryhiella salina and Mucor sp. The cultures were filtered through ignited Gelman type A glass fiber filters and the fatty acid composition determined by the same method as that employed for the natural retained organic matter. Thin-layer chromatography (t.1.c.) procedures for the isolation and purification of the fatty acid methyl ester samples were as previously described for retained organic matter (Schultz & Quinn, 1972). Gas-liquid chromatography (g.1.c.) was carried out on a HewlettPackard Model 5750 flame ionization gas chromatograph. The instrument was equipped
Organic detritus from Spartina alterniflora
181
with a polar column (I 5% stabilized DEGS on Gas Chrom P, 80/1oo mesh) and a non-polar column (2% Apiezon L on Anakrom Q, go/100 mesh). The columns used were 2.1 m by 32 mm o.d. (2.2 mm i.d.) stainless steel and were operated at 180 “C (polar) and 210 “C (non-polar), with nitrogen carrier gas (flow of 35-50 ml/min at 40-50 lb/in2 g inlet pressure). Fatty acid identifications were made on both columns by comparison of relative retention times of sample methyl ester peaks to those of authentic methyl ester standards and by coinjection of these standards with the sample. Quantitative values agreed before and after hydrogenation using Adam’s catalyst (PtO,). Peak areas of sample methyl ester peaks on the polar column were compared to the area of the internal standard peak (methyl heptadecanoate) to quantitate the results. The areaswere calculated by multiplying peak height by width at half-peak height. Quantitative results with National Heart Institute Fatty Acid Methyl Ester Standard mixture D agreed with the stated composition data with a relative error of less than 5qlo for major components (> 10% of total mixture) and less than 10% for minor components (
IS2
D. M. Schultz
M-7.
G. Quinn
Results The fatty acid composition of Spartim altemi~ora, from Bissel Cove, Narragansett Bay, Rhode Island, determined monthly from January, 1971 to December, 1971, is given in Table I. The fatty acid composition of natural retained organic matter, collected at both ebb and flood tides from Bissel Cove, for the same period is given in Tables 2 and 3, respectively. The three laboratory model studies are based on an aerobic system with S. &ernijoru, an anaerobic system with S. ulternifloru and an anaerobic system with extracted S. ulterni$oru. The fatty acid composition for the retained organic matter produced in these model studies are reported in: Table 4 (aerobic, S. uZterniJoru), Table 5 (anaerobic, S. uZterniJoru) and Table 6 (anaerobic, extracted S. uhrnijloru). The results of the field model studies are presented in Table 7 (5’. uZternifZoru) and Table 8 (extracted S. ulterniJoru). The fatty acid composition of the three fungi species isolated from S. ulterniforu are presented in Table 9. TABLE I. Bissel Cove, Narragansett Bay, Rhode Island. Fatty acid weight percentages of Spartina aZterniflo~u (stems and leaves)
Fatty acid“
Jan.
Feb.
Mar.
April
May
14 :o ‘Sb 16 : o
2’9 0.5 19’4 5.4 0.8 5.1 28.3 24’3 7.9
4.8 1.4 29.1 5.4 0’7 8.9 22.7 12’9 6.7 2.5 5.0
3.4 2.4 19’4 6.0 0.8 7.1
2.7 0’9 18.9 5’0 0.7 6.4 22.9 25’45 8. 4.6 3.9
0.9 0.7
16
: I
16 18 18 18 18
: : : : :
20
:o
22
:o
2 0 I 23
2.9 2.7
21’0 22'0 9.0
3.2 5.6
25’1
n.d.’ 0.3 1.8 6.3 ;I’; .
1971 June July 0.8 1.3 20.5
n.d. 0.6 2.1 4.5 y2.z .
1.5
1.3
n.d.
n.d.
Aug.
Sept.
Oct.
Nov.
Dec.
1'2
0.9
1.6 24’4 n.d. 0.7 2.1 4’0 2::;
1.1
1.6 0.4
1.9 n.d. 24.1 2.2
2.2 2.1 21.4 3.0 0.7 3’4 11.7
2.4 0’9
1.6 n.d.
25.6 1.7 tr 2.6 3’7
22'1
;;.z 2.2
Ti.82 1.6
1.0
2'0
1.2 0.4 2.6 6.0
1’1
3.1 7.8
22'0
4’2 0.6 4.8 22.4
25’5 J;‘; I’5 0.8
j;‘; '
11.8
2.5
2'0
1’0
3.6
Total pg/mg;” (SD.)
’ All fatty acids given by normal carbon chain length: number of double bonds. b Consists of undifferentiated species, including both normal and branched (iso and anteiso) r5-carbon acids and a small amount of 14 : I fatty acid. c n.d. =None detected. d pg fatty acid/mg dry weight Spurtim ulterniflo~u (standard deviation).
The monthly survey of S. ulternifloru stems and leaves (Table I) indicates two major trends. (i) The live (May-October) marsh grass is higher in fatty acid concentration (approximately 3-8 pg fatty acid/mg dry wt grass) than the dead grass (December-April, I-Z pg fatty acid/ mg dry wt grass). (ii) Although the fatty acid weight percentage composition generally agrees with published values (Maurer & Parker, 1967; Jeffries, 1972), there are rather drastic seasonal differences in the fatty acid composition between the live and dead S. ulttmi’ru leaves and stems. Among the major compositional differences are the decrease in 18 : 3 fatty acid from an average of 43.4% in the live samples to an average of 8.8% in the dead
Organic detritus from
Spartina
alternitlora
I83
grass. There is also a change in the amount of 18 : I acid percent, from an average of 5.4% (live) to 23’5% (dead). Similar trends have been reported by Jeffries (1972) for samples taken in February and June, 1970. TABLE z. Bissel Cove, Narragansett Bay, Rhode Island. Fatty acid weight percentages of natural retained organic matter-ebb tide
1971” June July
Aug.
Sept.
16.3 77
29.1
19.9
16.9
4.0
4.5
2.8 29'1 30'2 3'3
5.8 47'7 6.9 I'3
1.6
IO’I
4'4
4'2
3'2 36.1 17.0 0.8 2'7 4'3
5.3 3.6
tr.c n.d.
2’9 n.d.
3.9 35'5 22'1 1.9 2'0 8.2 2'0
6.7 2.6 43'0
Fatty acid
Jan.
Feb.
Mar.
May
I4 :o IS* 15 : 0 16 : o
17’9
8.8 3.6 2.5 39'9 33'9 I'9 2.4 5'5 1.8
15.1 47
16 : I 16 :2 18
: 0
18 : I 18
: 2
18
:
3
Total l.tg/mg;” (SD.) mg/le (S.D.)
2.6 2.4 38.6 19.1 3'3 3.2 2'1 8.8 2.1
n.d.
n.d.
10’0
Oct.
Nov.
Dec.
8.1
9.9 4.0
10.4 3.1
12.6
1.9 3.2 49'2 8.7
2.6 36.8
n.d.
3.5 3'1 5'3
2.5 9'4
15.8 0.4
5'3
4.3
3.7
12.7
4.6 16.6 2.6 6.7
2.9 34'9 11.2
2.4 9.2 14'9 6.2 4'9
1.8 I'5 34'9 14.5 2'1 25 13.2 7'0
10’0
(::;)
(t.:)
(2)
(:.i)
(E)
(:*I)
(:I;)
(2)
(2)
(;‘::)
(T::)
(2;)
(I”.;,
(g)
;“,p,,
(8::)
;::;,
;:.:,
(X.i)
(2)
(2)
(2)
’ April sample lost in analytical procedure. b Consists of undifferentiated species, including branched anteiso and iso, and a small amount of I4 : I fatty acid. c tr. =trace. d fig fatty acid/mg dry wt retained matter. e mg dry wt retained matter/liter Bissel Cove water.
I 5-carbon
fatty
acids,
TABLE 3. Bissel Cove, Narragansett Bay, Rhode Island. Fatty acid weight percentages of natural retained organic matter-flood tide
1971"
Fatty acid
Jan.
Feb.
Mar.
May
June
July
Aug.
Sept.
Oct.
Nov.
Dec.
14 :o 19 15 :o
13'9 2.1 1'4
14'5 3.8 2.3
14'9
15.8
26.7 2.6
22.8 3.2
12'1 6.7
4.6 2.9
12.9 3.8
8.8
11.7
1.6
2.0
2.8
6.9 4.5
16 16 16 18 18
36.1
32'5 24'5
32.1
40'5
12.7 1.7
6.2
14.8
4'7
3'4
1’1
2’1
: : : : :
o I 2 o I
18 :2
18 : 3 Total pg/mgC (S.D.) w/l* (S.D.)
1.8
1.8
1.8
3.0
36.1 2.3
2.4 4'2
4'7 7'3
2'2 3.8
9'2 7.4
25'4 27' I I'4 I'2 7.6
23
4.2 3.2
3.1 0.9
I'3 tr.
2.8 3'4
6.8 tr.
1'2 1.7
21.8
13.7
7.8
',"';)
(1.5)
(;.;I
(;:;)
(1.1)
2.6 (0.4)
(0.6)
(z.i)
(1.1)
(1.3)
(i.:)
;T:t)
30.8
6.2
5.5
12.2
15.8 2'2 6.1 15'2
6.3 8.9
i:i 14.1
2.1
4.2 30'3 13.5
3'2 15'4 1.6
6.7 9'4 6.8
7.6
(0.0)
(3.2)
(0.1)
(07)
(2.8)
;z.z)
(:I:)
;y:z)
(t.2)
(i:i)
’ April sample lost in analytical procedure. b Consists of undifferentiated species, including branched and iso, and a small amount of 14 : I fatty acid. c pg fatty acid/mg dry wt retained matter. d mg dry wt retained matter/liter of Bissel Cove water.
2'2 3'0
15-carbon acids, anteiso
D. M. Schultz &f J. G. Quinn
184
The May sample was the first of the new growth and the frost did not kill the grass until the end of October. However, the November sample, although taken after the first frost, still reflected the fatty acid pattern of the live grass more than that of the dead grass. The fatty acid concentration (4.2 pg/mg) was still high and the weight percent of the 18 : 3 acid has only dropped to 33.2% with that of the 18 : I acid (11.7%) being intermediate between the live and dead grass. TABLE 4. Fatty acid weight percentages of retained uZternifIora aerobic laboratory model system
organic matter from Spartina
Weeks Fatty acid 14 15 I5 16 16 16
:o
18
:o
I
2
2’9 0.5
3.1 8.1
2.1 8.2
tr.
:o :o : I
19’4 5’4
: 2
0.8
2.8
I'2
17’4 14’0
2’7
23’5 12.4 0.6 4.0 34.6 7.1 I.4 2.4
1'2
9’9
5’1 28.3 24’3
I8 : I I8 :2 18 : 3’ 22
0
IO.8
:o
Total pg/mgb
1.5 2’4 43’0 2.9 2'1
2.
3
5
6
8
8.0 8.8 3’4 23.2 14’5
2.9 10.9 I’7 22.5 19’5
2.6 8.4 I’3
g:;:
21'1
22.4
I.5
;:;
::i
3’8
22’9 2.8 3.1 3’5
26.4 3.6 2.2 3’2
29.6 2.7 I.9 4’9
5’9
5’1
5’2
IO 5.2
12 6.9
8.1 I’5 25.1 18.3 I.7 6.2 20.5 2.2
1’1 23.0 18.7
;:;
I’3 20.9 18.7 2'1
I7 7’5 8.4 2'2
18.1 20’2
;:;
5’9 25.8 I’9 2.8 5’7
23’4 3’0
. :.:
25.1 4.2 4’7 7’0
3’3
2’4
32
3.2
;:;
a Consists of both I8 : 3 and 20 : o fatty acids. b pg fatty acid/mg dry wt retained matter. TABLE 5. Fatty acid weight percentages of retained organic model system
matter
from
Spartim
aZternifIoru anaerobic laboratory
Weeks Fatty acid 14:o I5 ‘5 :o 16 : o 16 : I 16 : 2 18:o 18 : I
0
3
6
2.6
4’3 10'0
4’0 13’9 3’1 23-3 20.9 3’0 5’9 22.6
I’5 1.6 19.5 5’3 I '0
6.9
18:2
32'4 I9.I
18 : 3
IO'2
20
:o
Total pg/mg’ (S.D.)
tr.
(2)
2.6 21'1
29.0 2’7 2’5 24'7 1'3 0.8
;:; (0.3)
I'3 1'0
IO
4’4
5’4
14.8
IO.8
4’2
2’3 47‘4 7‘4 5’4 10.4 8.7
21'0
21.5 7’1 2.8 17.8 2'1 I.3
I’I . (&)
17
1'1
;:::,
n.d. n.d. z-3 (2)
LIpg fatty acid/mg dry weight retained matter.
The dead grass remains standing through the winter until the spring thaw. It is then sheared off and transported from the cove into the bay in fairly large rafts by tidal action (Nixon & Oviatt, 1973).However, the grass in these rafts is largely intact and one would not expect a very abrupt change in the suspended matter content of the water. In addition, large
Organic detritus from Spartina
alterniflora
185
rafts are stranded on the shores of the cove and are slowly removed by the tidal action, thereby providing a source of suspended organic matter over an extended period of time. In order to determine what percentage of the fatty acids in the retained organic matter is contributed by dissolved fatty acids retained by the filter, an experiment was conducted using two filters, one on top of the other. After Bissel Cove water was passed through the filters, both filters and the filtrate were analysed for fatty acids. Based on the amount of fatty acids retained by the second filter in this study, approximately zoo,&of the values reported in this work result from retention of dissolved fatty acids by the Gelman A filter. TABLE 6. Fatty acid weight percentages of retained organic matter model system
from extracted’
Spartim alternifora anaerobic laboratory Weeks Fatty acid 14 :o
2’4
15
I5 16 16 16 18 18 18 18
:3
20
:o
8
12
16
“5
7’7 8.0 4.2 32.2 IO.4
2'2
4
:o :o : I :2 :o : I : 2
Total ug/mgb
11'5
II'2
1’2 28.5 8.7 6.2 7’0 15’7 18.9 n.d. n.d. 6.0
1.6 19’5 9.5 2’7 4.8 29.2 ZO'I
nd. n.d. 4’0
;:; 16.9 5’3 n.d. 5.1 10.9
6.0 0.9 30’4 13.1 2’7 7’4 12.5 25.0 nd. n.d. 22.6
‘Extracted by repeated treatment with organic material. b pg fatty acid/mg dry wt retained matter.
solvents to remove
TABLE 7. Fatty acid weight percentages of retained organic matter ~Zternifloru field model system (9 June to 17 November 1971)
Fatty acid :o
o
1
z
3
4
2.9 1’4 1’0 19’9 7’2 0.5 7’3 15.6
4.1 4’7 3’3 26.9
4.6 7’4 4’0 27.6 15’5
5.4 15.8 4.2 22.6 14’3 2.8 5’9 16.7 2’9 1.2 2.6 6.0 ’ (i.T)
14 ‘5 I.5 16 16 16 18 18 18 18
:0 :o : 1 :2 :o : 1 :2 :3
20 22
:o :o
7.2 7’7
Total pg/mg’ (SD.)
(0.3)
20'5 8.2
2.2
3.4 3-3 3’4
I% 3’0
5.1 7’5 5’5 25.0 15’4 2.8 5’0 22.6 3’0 I.9 1.8 4’3
6.7 (0.8)
. (L.:)
6.2 (0.5)
11’0 1.0
1.2
6.6 28.2
5’3 26.7
4'1
. 2.1
5
Weeks 6
z::
2.2
11.4 4’9 n. d . 3.6 n.d.
12.5 n.d. n.d. n.d. n.d.
5.0 4.1 13’3 26.2 18.0 6.2 2.8 15.6 1.9 “3 1.2 4.4
6.0 (0.9)
. (‘0.:)
6.2 (0.9)
5.7 22'2
6.1 25.9 IO.9
7.8 6.6 16.6 30.1 ‘4’7 9.5
8
u pg fatty acid/mg dry weight retained matter.
natural
lipid
from Spartim
14
18
23
5.8 9’4 4.2 26. I 16.5 7.5 3.4 21.8 2.0 1.7 nfc? .
7.2 7’5 5.1 30.8 18.4 6.9
2.9 4.4 tr. 4.4
7.1 10’2 7.6 26.0 16.2 4’9 3.5 16.9 1.7 2.6 n.d. 3.3
2.2
6.5 12.6 3.6 28.8 21.9 4.4 14 16.3 0.6 1’7 n.d. 1’7
8.0 (1.3)
(:I:)
(i.i)
(t.z)
(i.:)
IO
12
5.1 4.5 4.8 44.8 II'0
3.8 3.3 II'2
2.0
17.2 0.9 1.8 n.d.
186
D. M. Schultz &J. G. Quinn
The natural retained organic matter showed little difference in the fatty acid compositions between the ebb (Table 2) and flood tides (Table 3). For example, the 16 : o fatty acid (&S.D.) averaged 38*7-&5+9% in the ebb tide samples and 33+3&5.8% in the flood tide samples, and the total Is-carbon acids (anteiso, iso and normal) were 7-I&2*7% (ebb) W~YSUS 6*2f2*5% (flood). The 14 : o acid average was 15*0&6~1‘J(~(ebb) and 14*4&6*1’7~ (flood) and the 18 : I average was 8*0&-4*9% (ebb) and 9*1&3*7% (flood). However, the total fatty acid concentration (pg FA/mg dry wt retained matter) of the flood tide was significantly higher (at the 95% confidence level) than that of the ebb tide in 7 of the II samples (ebb tide-average 5*0&1-3 pg/mg, flood tide-average 7*9& 1-6 l.tg/mg). In addition, the data in Tables 2 and 3 indicate a general flux of retained matter outward from the marsh into the bay (significant at the 90% confidence level in 8 of the II samples). The values of mg dry weight retained matter/liter of Bissel Cove water are ebb tide-average 8.of1.7 mg/l and flood tide-average 6.0% I-O mg/l. The ebb tide appears to be transporting suspended matter from the cove into the bay which, however, is lower in fatty acid concentration than that already in the bay. This might indicate that the suspended matter transported from the cove to the bay has less microbial growth associated with it and may be recently synthesized detritus produced from the dead marsh grass. TABLE 8. Fatty acid weight percentages of retained organic matter from extracted” Spartim alterniflora field model system (3 August to z November 1972) Weeks Fatty acid 14 :0 I5 15 16 16 16
:o :o : I :2
18 : o 18 : I 18 : 2 18 : 3 20 : 0 Total l.tg/mgb
3
9
13
8.0
5’4
7’0
32.1
13.0 6.7 26.2
24’4 4.9 1.6 15’3
23’5 2.8 I’9 20.7
8.1 9’4 27.0 20’7
n.d. n.d. n.d.
n.d. n.d. n.d.
9’7
3’1
a::
2.7 3’0 17.1 1.6
I’S 2’0 2’2
a Extracted by repeated treatment with organic solvents to remove natural material. b l,tg fatty acids/mg dry wt retained matter.
lipid
The effect of phytoplankton blooms in the bay on the fatty acid composition of retained organic matter appears to be fairly small, since the amount of detrital material from plant decomposition is much larger than the amount of living phytoplankton contained in 2-4 liters of water from the mouth of the cove. Since there were none of the marine polyunsaturated acids (such as 20 : 5 and 22 : 6; Jeffries, 1970) found in the retained organic matter samples, this observation seems valid, although it must be re-emphasized that these polyunsaturated acids would probably be masked by the large amount of terrestrial suspended matter present in the water in Bissel Cove. There were also large amounts of 18 : 3 found in several of the samples of retained organic matter (e.g. September) which can not be explained at this time.
Organic
detritus from
Spartina alterniflora
187
The fatty acid composition of marsh grass (Table I) differs from that of the natural retained organic matter (Tables 2 and 3) in several major areas. The marsh grass generally has more of the unsaturated acids 18 : I, 18 : 2 and 18 : 3 than is found in the retained organic matter. Both the 14 : o and 16 : o acids showed lower trends in the marsh grass than in the retained organic matter. In addition, the unsaturated 16 : I acid and the total Is-carbon acids tend to be much lower in the marsh grass than in the retained organic matter. The relatively long chain saturated acids 20 : o and 22 : o were present in the marsh grass but not detected in the retained organic matter. TABLE
9. Fatty
acid weight
percentages
of fungi
species isolated
from
Spartinn
alternifEora
Fatty Acid I4 : 0 15 *5 16 16 16
:o : o : I : z
18
:o
18 : I 18
:2
18 : 3
Sphaerulina pedicellata
Dendryphiella salina
Mucor species
0.9 3.8
n.d.
n.d. n. d n.d.
n.d. n.d.
20.4
30’2
26.8
4.6 0.9 7’0 27’4 33’0
3’4 n.d.
n.d.
6.4 23.6
40’9
2.0
36.4
n.d.
0.8
1'2
12'0
18.3 n.d.
The procedure for differentiating esterified and free fatty acids was performed on a natural retained organic matter sample collected at ebb tide and provided additional information concerning the nature of the fatty acids present in retained organic matter. Approximately 60% of the fatty acids were present in an esterified form in this sample, compared to >99% present in the esterified form in a sample of dead 5’. aZterniJora. This procedure may be useful in tracing the pathways of organic matter conversion in estuarine environments by differentiating between various sources and processes. Additional studies using this procedure are now in progress. The model studies were designed to compare the natural suspended matter from Bissel Cove with material produced by allowing dead S. altern$ora to decompose under laboratory and field conditions. The studies using extracted marsh grass were performed in an attempt to see if microbial growth on fatty acid-free particles was the most important source of fatty acids in detritus formation. The model studies with the S. aZterni$ora revealed several interesting trends. The aerobic laboratory model system (Table 4) produced material in which the weight percent of the saturated acids 14 : o and 22 : o generally increased over the 17 week period. The 16 : o, 18 : o and 18 : I acids were variable over the time interval, but remained essentially constant. The total Is-carbon acids showed a very marked increase over the time interval, going from o-50/Oinitially to 10*60/Oafter 17 weeks. Among the unsaturated acids, 16 : I increased from 5.4% to 20*2%, 16 : 2 showed a slight increase and both 18 : 2 and 18 : 3 (20 : o) decreased markedly (35.1% total initially to 4.7% total after 17 weeks). Under anaerobic conditions (Table 5), similar trends were observed with the exceptions that the 16 : o acid increased between the tenth and seventeenth week, the 16 : I acid generally increased to IO weeks and decreased at 17 weeks, 16 : 2 increased substantially
188
D. M. Schultz &f J. G. Quinn
1.0 to 7.1~/~) to IO weeks and the 18 : I acid generally decreased over the r7-week period. In addition, the saturated 22 : o acid was not detected in any of these samples. At the end of 17 weeks, the fatty acid composition of the retained organic matter produced by both systems showed values that were considerably different from the initial S. alterniflora. The aerobic system had a high concentration of 16 : I and 18 : I, whereas both these acids were rather low in the anaerobic system and 16 : o and 18 : o acids were higher in the anaerobic than aerobic system. The total concentration of acids (pg/mg) increased from 1.2 to 3.2 pg/mg, with a maximum at I week for the aerobic and increased from 1.5 to 7.6 pg/mg (maximum at IO weeks), for the anaerobic system. These values, 3.2 and 7.6 pg/mg, are similar to most of the values reported in Tables 2 and 3 for natural retained matter, especially those of the ebb tide samples (2-1 to 8.5 pg/mg) which might reflect values of newly synthesized detritus transported from cove to bay as previously described. The trends observed in the field study (Table 7) were similar to those observed for the laboratory studies with most values closer to those of the aerobic laboratory system. Although the 16 : o acid generally increased over the r8-week period, the other saturated acids (18 : o, 20 : o, 22 : o) generally decreased over the time interval. The total concentration of fatty acids increased after I week and then remained essentially constant through 18 weeks. The value (6.3 pg/mg) at I 8 weeks resembled the anaerobic study more than the aerobic study (7.6 and 3.2 pg/mg, respectively) after 17 weeks and also agree with values for ebb tide (Table 2). The trends for the individual fatty acids over the r8-week period generally held after 23 weeks, although the fatty acid concentration decreased during this period. Thus, the available evidence from these model studies indicates the following. (i) Both aerobic and anaerobic processesmay be important in the formation of detritus. (ii) Although the percentage of the 18 : 3 acid decreased under both aerobic and anaerobic conditions, the anaerobic process appears to be important in producing levels of 18 : I and 18 : 2 acids similar to those found for natural retained organic matter. This may be the starting point of suspended matter formation in the marsh, since the sediment/water interface is anaerobic during much of the time in which detrital formation would be occurring. (iii) The total fatty acid concentration @g FA/mg dry wt retained matter) of natural and model retained matter was very similar. (iv) The IS-carbon acids may be the products of microbial activity (Parker et al., 1967) and in a closed system accumulate to a very high level. (v) The concentration of fatty acids generally increases over time, with qualitative changes readily apparent. In order to determine whether the fatty acids being measured were from S. alternijoru itself or bacteria and other micro-organisms living on the particles, a laboratory model system (Table 6) and a field model (Table 8) were investigated using S. ulternifloru from which the fatty acids had been extracted. The intent was to use the extracted grass as a solid support, providing glucose as a carbon source and measure qualitative and quantitative changes in the fatty acids. The initial fatty acid concentration of the grass was about 0.1 pg FA/mg retained matter. After 16 weeks the laboratory model retained matter had a fatty acid concentration of 22.6 pg/mg (Table 6) (aft er correcting for the controls), giving a fatty acid increase of about 200 times over the initial value (22*6/o-1). Since small particles which break loose from the dead grass had essentially the same fatty acid composition and concentration as the intact grass, the increase must be attributed to some type of microbial growth. The fatty acid composition of the laboratory retained organic matter tended to be higher in the unsaturated acids 18 : I and 18 : 2 than the natural retained organic matter but the levels of 16 : 0, 16 : I, 16 : 2, 18 : o and total Is-carbon acids were very similar to the natural
Organic detritus from Spartina
189
alterniflora
retained matter. However, the 14 : o and 18 : 3 acids both tended to be lower in the laboratory retained matter than in the natural samples. In the field study with extracted marsh grass (Table 8), an increase was observed in the fatty acid concentration of the retained matter (about 6 times) after 13 weeks (from 0.3 pg/mg initially to 2.2 pg/mg). The fatty acid composition of the retained organic matter produced in this study showed similar trends to the laboratory study, with the exception that the 14 : o and 18 : 3 fatty acid weight percentages were now also similar to that found in the natural retained organic matter and the 18 : I and 18 : 2 acids were lower than those in the laboratory study. The fatty acid composition of this field study was very similar to that found after 18 weeks in the field study using the non-extracted S. alterniflora (Table 7) and both gave a fatty acid composition and concentration very similar to the natural retained organic matter from ebb tide. Both of these models gave indications that the entire fatty acid concentration and composition of the natural retained organic matter can be accounted for by the activity of associated micro-organisms. The fatty acid composition of the fungi (Table 9) all contain fairly large amounts of 16 : o, 18 : o, 18 : I and 18 : 2, but only Sphaerulina pedicellata contains any IS-carbon acids. This fungus is one of the more abundant fungi isolated from the Bissel Cove salt marsh (Gessner, 1972) and appears to initiate natural fungal colonization of Spartina alternijlora (Gessner et al., 1972). Teal (1962) estimated that in the Georgia marshland, bacterial activities involved in degradation of S. alterniflora accounted for 59% of available plant substrate. However, the ‘bacterial’ activities were not subdivided into bacteria and fungi. The role of fungi in the degradation of plant material has been studied in a fresh water environment and Kaushik & Hynes (1971) state that fungi are more important than bacteria in the initial stages of the degradative process. Some of the acids present in the retained organic matter formed in the model studies could be a result of the presence of these or other fungi species, although only Sph. pedicellata contains any of the Is-carbon acids. It appears that the fungi alone cannot totally account for the fatty acid composition of any of the model studies or the natural retained organic matter. Marine bacteria are high (up to 57%) in the branched (iso and anteiso) Is-carbon acids (Parker et aZ., 1967) and a combination of bacteria, fungi and other micro-organisms probably contributes to the fatty acid composition of the natural retained organic matter. There was fairly good agreement in the fatty acid composition and concentration for retained organic matter produced in laboratory and field model systems and natural retained organic matter. The model systems indicated that the actual formation of the natural suspended organic matter is a very complex process which probably involves both aerobic and anaerobic reactions. In addition, natural organic detritus is a very heterogeneous mixture of material in varying stages of decay and containing various amounts and types of micro-organisms. Acknowledgements This investigation was supported by the National Sea Grant Program (04-3-158-3). We thank Dr H. Perry Jeffries for reviewing the manuscript and Mr Robert V. Gessner for providing the fungi cultures. References Ast., H. J. 1963 Inadvertant
isomerization tical Chemistry 35, x539-15+0.
of polyunsaturated
acids during
ester preparation.
Analy-
190
D. M. Schultz &f J. G. Quinn
Burkholder, P. R. & Bornside G. H. 1957 Decomposition of marsh grass by aerobic marine bacteria. Bulletin of the Torrey Botanical Club 84, 366-383. Darnell, R. M. 1967~ The organic detritus problem. In Estuaries (Lauff, G. H., ed.). American Association for the Advancement of Science, Washington. pp. 374-375. Darnell, R. M. 19676 Organic detritus in relation to the estuarine ecosystem. In Estuaries (Lauff, G. H., ed.) American Association for the Advancement of Science, Washington, pp. 376-382. Gessner, R. 1972 The fungi associated with Spartina alterniflora and other salt marsh plants from southern Rhode Island. Masters thesis, Univ. of Rhode Island. pp. I-IZZ. Gessner, R. V., Goes, R. D. & Sieburth, J. McN. 1972 The fungal microcosm of the internodes of Spartina alterniflora. Marine Biology 16, 269-273. Gunstone, F. D. 1967 An Introduction to the Chemistry and Biochemistry of Fatty Acids and their Glycerides. Chapman and Hall, Ltd, London. pp. 1-209. Heald, E. J. 1971 The production of organic detritus in a south Florida estuary. Sea Grant Technical Bulletin Number 6 Univ. of Miami. pp. I-I IO. Herb, S. F. & Martin, V. G. 1970 How good are analyses of oil by g.1.c. Journal of the American Oil Chemists Society 47, 415-42 I. Horning, E. C., Ahrens, E. H. Jr, Lipsky, S. R., Mattson, F. II., Mead, J. I;., Turner, D. A. & Goldwater, W. H. 1964 Quantitative analysis of fatty acids by gas-liquid chromatography. Journal of Lipid Research 5, 20-27. fatty acids. Limnology Jeffries, H. P. 1970 Seasonal composition of temperate plankton communities: and Oceanography 15,419-426. Jeffries, H. P. 1972 Fatty-acid ecology of a tidal marsh. Limnology and Oceatzogvuphy 17, 433-440. Kaushik, N. K. & Hynes, H. B. N. 1971 The fate of dead leaves that fall into streams. Archiw FZIY Hydrobiologie 68,465-5 I 5. Mann, K. H. 1972 Macrophyte production and detritus food chains in coastal waters. Symposium on ‘Detritus and its ecological role in aquatic ecosystems’ zznd-26th May 1972, Pallanza, p. I-57. Maurer, L. G. & Parker, P. L. 1967 Fatty acids in sea grasses and marsh plants. Contributions in Marine ,%ie?nCe 4, 113-119. Newell, R. 1965 The role of detritus in the nutrition of two marine deposit feeders, the prosobranch Hydrobia ulvae and the bivalve Macoma balthica. Proceedings of the Zoological Society of London 114, (I), 25-45. Nixon, S. W. & Oviatt, C. A. 1973. Ecology of a New England Salt Marsh Ecological Monographs. In press. Odum, E. P. & de la Cruz, A. A. 1967 Particulate organic detritus in a Georgia salt marsh estuarine ecosystem In Estuaries (Lauff, G. H., ed.) American Association for the Advancement of Science, Washington. pp. 383-388. Odum, W. E. 1968 The ecological significance of fine particle selection by the striped mullet Mugil cephalus. Limnology and Oceanography 13, 92-98. Odum, W. E. 1g70a. Pathways of energy flow in a south Florida estuary. Ph.D. dissertation, Univ. of Miami. 126-136. Odum, W. E. Ig7ob Utilization of direct grazing and plant detritus food chains by the striped mullet Mu&l cebhalus. In Marine Food Chains (Steele, J. H., ed.) Univ. of Calif. Press, Calif. pp. 222-240. Parker, ‘I’. L:, Van Baalen, C. & Maurer, L. 1967~Fatty acids in eleven species of blue-green algae: geochemical significance. Science 155, 707-708. Quinn, J. G. & Meyers, P. A. 1971 Retention of dissolved organic acids in sea water by various filters. Limnology and Oceanography 16, I 29-13 I. Schultz, D. M. & Quinn, J. G. 1972 Fatty acids in surface particulate matter from the North Atlantic. Journal of the Fisheries Research Board of Canada 29, 1482-1486. Teal, J. M. 1962 Energy flow in the salt marsh ecosystem of Georgia. Ecology 43, 614-624. Ustach, J. F. 1969 The decomposition of Spartina aZterni$ora. Masters thesis, North Carolina State University. pp. 1-26.