Microbial production of an estuarine mudflat

Estuarine and Coastal Marine Science (1978) 7,

185-195

Microbial Production of an Estuarine Mudflat

I. IQ.Joint NERC Institute for Marine Environmental Research, Prospect Place, Plymouth PLI JDH, U.K. Received

12

April

1977

and in revised form

Keywords: microflora; production;

I4

October

heterotrophs;

I977

mudflats; water columns

England coast The components of microbial production of an intertidal mud flat in the R. Lynher, S.W. England, were studied from January 1974 to December 1974. Measurements were made of primary production on the sediment surface and in the water column, and the seasonal changes in numbers of aerobic heterotrophs and carbon and nitrogen content of the surface sediment were measured. The annual primary production was 143 g C m-* for the sediment and 81.7 g C me2 for the water column. Primary production on the sediment surface ceased when the mudflat was tidally flooded. Successive peaks of chlorophyll a in the water column were associated with phytoplankton of different cell sizes. Functional chlorophyll a was found at depths of greater than IO cm in the sediment. Attempts to measure the heterotrophic potential of the microbial populations were equivocal but suggested that heterotrophic microbes were not very important in the turn-over of carbon on this mudflat. The source of carbon is assumed to be autochthonous and the capacity for the measured primary production to support the macrofauna production is discussed.

Introduction Intertidal mudflats are areas of high macrofauna production (Warwick & Price, Ig75), but there have been few studies to determine if this high level of production is mainly the result

of carbon fixation by the microflora of the sediment surface or of planktonic primary production in the water column: relatively high rates of primary production might be expected by planktonic plant cells because the shallow water they inhabit increases in temperature quickly in the summer and is continually enriched by inorganic nutrients released from the bottom sediments. The purposes of this investigation, which forms part of a study of the energy relationships of an intertidal mudflat, were threefold : to quantify the rate of production of both the microflora of the sediments and the phytoplankton of the water column; to determine the rate of turnover of carbon by heterotrophic microbes, and to compare’these rates with the macrofauna production

measured on the same mudflat

by Warwick

& Price (1975). The investigation

was performed by monthly sampling at a single site during one year.

Methods The study site and its sediment characteristics

have been described previously

(Warwick

&

Price, 1975). Production was measured, in situ, at the mid-tide level of the mudflat; all 1% 0302~~524/78/0801-0185

$02.00~0

0 (1978) Academic Press Inc. (London) Ltd.

186

I. R. Joint

samples were taken just after the site was exposed by the falling tide. Measurement of water column production was made on samples taken over the site 2 h before a high water which occurred near midday. Measurements of primary production at the sediment surface were made using a modification of the %I! method of Marshall et al. (1972). Corers, constructed from z-ml plastic syringes, were used to take ten replicate samples of the surface sediment, each 0.5 cm long, which were placed in small plastic cups of slightly larger diameter than the cores which, because the mud surface was very cohesive, maintained their structure during manipulation. The plastic cups were gently immersed in IOD ml seawater collected from the sampling site just before the mudflat was exposed ‘and filtered through a o.45-prn Millipore filter before use. Five samples were incubated in daylight ‘on the mudflat surface well away from any shading effects, each sample having been inoculated with I pCi Na, 14COa; five replicate samples were also incubated in the dark. Two additional samples were filtered immediately after addition of 14C, as controls. Samples were routinely incubated for 4 h but occasional time-course experiments, taking samples at 30 min intervals, were done; both methods gave the same rate of CO, fixation at all times of the year. At the end of the experiment, each bottle was shaken vigorously to suspend the sediment, filtered through o’45-pm Millipore filters which were then stored frozen. The filters were subsequently dried to remove any unfixed 14C and ground up with 3 ml a-methoxyethanol to produce a slurry, aliquots of which were added to a 4% suspension of Cab-0-Sil in toluene scintillant to produce a thixotropic gel with the sediment particles evenly dispersed. Each sample was then counted in a liquid scintillation spectrometer; the counting efficiency was determined by the external standard, channels ratio method. Quenching by the suspended sediment, as determined by internal standardization, was not significant if the total weight of sediment did not exceed 20 mg dry weight in each aliquot. Measurements of primary production in the water column were made on water samples suspended at the surface and at 0.5 m from a raft, in mid-channel near the site, which was constructed to minimize shading effects. Each IOO ml sample was incubated with I $i Na, 14C0, for 4 h filtered through a o*45-pm Millipore filter which was then stored,frozen. The filters were subsequently dried to remove unfixed 14C, dissolved in a-methoxyethanol and toluene scintillant and counted in a liquid scintillation spectrometer. Replicate water samples were filtered through GFC glass fibre filters extracted with 90% acetone and analysed for photosynthetic pigments by the method of Strickland & Parsons (1968). Heterotrophic potential was measured by the [t4C]-glucose method of Crawford et al. (1973). Undisturbed cores, and slurries, of surface sediment from the upper 0.5 cm were incubated in 50 ml filtered seawater with varying concentrations of 14C-glucose for I h: the incorporation of 14C into particulate material and the production of 14C0, by respiration were measured. Sediment samples were treated in the same way as the primary production samples and counted in a liquid scintillation spectrometer. The production of 14C0, was measured by trapping the 14C0, produced by respiration on a filter paper moistened with phenylethylamine; the efficiency of recovery of CO, was measured by adding known amounts of Na, 14C0, to control flasks. Sediment cores up to 25 cm in length were taken by pressing a Perspex tube of 5 cm diameter into the sediment. The cores were frozen, cut into sections whilst still frozen and sub-samples were taken from the centre of each section to minimize contamination with surface sediment; surface sediment samples were taken in the same way as samples for primary production. The carbon and nitrogen content of the core samples was measured

Microbial

production of an estuavine mudfat

187

with a Perkin Elmer 240 CHN analyser, calibrated with acetanilide, sediment samples being dried, weighed and treated with hydrochloric acid to remove carbonates before analysis. Photosynthetic pigments were extracted with 90% acetone from weighed sediment samples and the absorption spectra measured in a Pye Unicam SPSooo recording spectrophotometer following the procedure of Strickland & Parsons (1968). Estimates of heterotrophic microbes were made by plating samples of sediment and water on marine medium 2216 (Zobell, 1941) made up with water of 25x0 salinity. Particle counts were made on water samples with a Coulter Counter model B with a model M volume converter; water samples were stored for up to 6 h in the dark before particle counts were made. Results Rates of primary production The seasonal rates of photosynthetic fixation of carbon on the mud surface and in the water column are shown in Figure I. The rate of photosynthesis of the microflora of the sediment surface increased rapidly during April to IOO mg C mW2h-l; this rate was maintained until August when it declined slowly to the winter value of IO mg C mV2 h-l. The summer increase in the rate of photosynthesis in the water column occurred later, probably because the seasonal increase in water temperature lagged behind that of the sediment surface: the temperature of the water in April (I 1-5 “C) was 4’7 “C less than that of the sediment surface after tidal exposure for 4 h. The coefficient of variation between monthly determinations of photosynthetic rate was 4.8% for the sediment and 16.8% for the water column.

1

Figure I. The rates of primary column (0) during rg74-

production

of the sediment

(0) and the water

Since the water over the tidally submerged site was relatively clear (Secchi disc reading of 2 m), some photosynthesis could have been expected on the sediment surface even when the mudflat was submerged. However, it was found that the rate of photosynthesis decreased rapidly as the mudflat was submerged and was not detectable, that is, was less than 0.25 mg C mm2 h-l, only 30 min after flooding. At this time, the water depth was less than 0.5 m and the light intensity at the sediment surface was 76% of that at the sea surface;

188

I. R. Joint

TABLE I. Calculated column

mean monthly

primary

production

Water column

Sediment

Mean rate production (mgCm-x h-r) January February March April May June July August

Hours exposed in daylight

Mean rate production (mgCm-a h-r) I.Za

115 96

201

228

21888

108

243 238 217

2624.4

23 800

3.8 43’7 42’3 33’1

20832

29'0

188 16;

11280 5610

141 157

3243

15’5 2.8 2.7 2.7

18

100° 96

60’

October November December

129

Mean calculated primary production (mgCme2 month-r) 645 1332 3114 23 115

5 9

34 23 13

“Estimated

148 I73

for the sediment and water

2041

1'2

0.9

Hours daylight 254 274 366 411 481

489 496 .?46 375 328

26.5 242

Mean calculated primary production (mgCm-a month-r) 305 330

329 1562 21019 20685

16 417 12 934 5812 918 716 653

value.

10% illumination was measured at a depth of z m. The integrated primary production for the year was therefore calculated only for those daylight hours when the mudflat was exposed. The calculated mean monthly production for the sediment and the water column are given in Table I : the integrated total production for 1974 was 143 g C m-2 for the sediment and 81.7 g C rnT2 for the water column.

Chlorophyll a content of the water column The seasonal cycle of chlorophyll a values is shown in Figure z; it contains two peaks of chlorophyll a, although the seasonal cycle of photosynthesis included only a single peak (Figure I). The first pigment peak occurred in May and a second, larger peak occurred in July; particle size measurements with a Coulter Counter suggest a different phytoplankton composition for the two chlorophyll a maxima. Figure 3, shows the size distribution of particles present in the water column; those present in March were less than IO pm in diameter and almost entirely comprised non-living particles brought into suspension during the winter. There were two sizes of particles present in May with mean diameters at 6 urn and 40 pm. Although no attempt was made to identify the phytoplankton present, microscopic examination of the particulate material showed the presence of short chains of small cells, but no large cells, and the particles of 40 urn counted by the Coulter Counter were probably such chains; there was no single dominant species present. In July, the maximum chlorophyll a concentration was associated with a broad spectrum of particles with diameters from 4 pm to 60 pm; microscopic examination showed a heterogeneous population of large cells, chains of small cells, and small cells. The October size distribution of particles, at a time of low chlorophyll a, was due largely to non-living particles, the diameter of particles resembling that present in March. Chlorophyll content of the sediment The seasonal cycle of the chlorophyll a content of the surface sediments is shown in Figure

Microbial production of an estucninemu&Tut

Figure 2. The concentration

of chlorophyll

a in the water column during

189

1974.

,+(a) and the increase in chlorophyll 4 during April coincided with the increased rate of photosynthesis shown in Figure I. The increase in standing stock of chlorophyll a between March and April was 39 pg g- 1 dry sediment, equivalent to an increase in biomass of x2.5 g C mW2, if a carbon to chlorophyll a ratio of 50 is assumed: the calculated photosynthetic production for the same period was 20 g C m-s. There was a decrease in the chlorophyll a content of the surface sediment during May but this increased again during

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19 March 1974

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Figure 3. The size distribution of particles in the water column, expressed as parts per million on a volume basis, at four times during 1974.

I. R. Joint

190

100 ~~~~~~~ P

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(d 1 Nitrogen

(c) Heterotrophs l8-

4,5-

6’.

E {

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4.0-

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JFMAMJ

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Figure 4. Measurements made on the surface 0.5 cm sediment. (a) Chlorophyll a, (b) Carbon content, (c) Numbers of aerobic heterotrophs, (d) Nitrogen content. The vertical bars are two standard errors.

July. The Lynher estuary is sheltered and the mudflat stable (Warwick & Price, 1975), so that the decrease in chlorophyll a content was probably not the result of disturbance by wave action, but rather due to the turnover of sediment by animal activity. Figure 5 shows the depth profile for chlorophyll a and phaeopigments at five intervals during the year. Negligible amounts of chlorophyll a, or of phaeopigments, were found at depths greater than 20 cm; the maximum chlorophyll a content of the upper 2 cm of sediment occurred in August but more than twice this concentration was found in the surface o-5 cm. Clearly, throughout the year, the microflora was concentrated at the sediment surface but was also transported into the sediment to considerable depths. Indeed, the core taken in June suggested a content was an accumulation of chlorophyll a at 12 cm; this increased chlorophyll associated with high values of phaeopigments suggesting the degradation of functional chlorophyll a at this depth. Carbon and nitrogen content of the sediment Figure 4(b) shows the carbon content of the surface 0.5 cm which exhibited a steady decline throughout the year with only a slight increase in August. The high carbon content of the surface sediment layer at the beginning of the year was probably the result of a bloom of Enteromorphu during the summer of 1973. Enteromorphu was not present during the year of this study: however, it was present in abundance in 1973 and again in 1975 when the maximum biomass was 95 g C me2 in early September; at the end of the 1975 bloom the Enteromorphu was not washed away in significant amounts, but buried in the sediment. If the same situation occurred at the end of the 1973 bloom, the Enteromorpha biomass could account for the elevated carbon content of the surface sediment in early 1974. The nitrogen content [Figure 4(d)] did not show the same decrease as the carbon in the sediments but there was an increase of I mg N g-l sediment during the summer.

Microbial production of an estuarine mudflat

Chlwophyllo(pg/g

191

dry sediment 1

6

6

6

IO

IO

IO 14 ~

21 Jun.‘74

22 Feb.‘74

6 Aug.74 Phoeopigments

IO

20

IO

(pg/g

2 Oct.‘74

2 Dec.‘74

2 Oct.‘74

2 Dec.‘74

dry sediment )

20

2 g c 2

6 IO 14 22 Feb.‘74

21 Jun.‘74

6 Aug.‘74

Figure 5. The concentration of chlorophyll a and phaeopigments, pigment per g dry sediment, in cores taken at 6 intervals during

expressed as pg 1974.

Production of heterotrophic microbes

4(c) shows the numbers of heterotrophic microbes present in the aerobic layer of sediment as estimated by growth on nutrient agar plates. No attempt was made to enumerate anaerobic or facultative microbes and the uptake and respiration of 14C-glucose was used as a measure of heterotrophic production. Because it was not possible to measure the natural glucose concentration of the water column or sediment the kinetic technique of Crawford et al. (1973) was adopted. The data were analysed by a modified Lineweaver-Burk plot of the reciprocal of rate of uptake of [14C]-glucose, or rate of production of 14COafrom [‘“Clglucose, against the added glucose concentration; only one of the ten experiments gave a correlation coefficient above the 95% probability level. Although duplicate samples at the same glucose concentration gave good reproducibility, the data did not obey MichaelisMenten kinetics: this was the case with both undisturbed sediment cores and slurries of aerobic sediment. The one acceptable set of data was obtained on 22 May and the calculated V max was 0.165 ng C rnB2 h-l. No other experiment gave acceptable kinetics and in the summer months the modified Lineweaver-Burk plot occasionally had a negative slope: it is unlikely that this resulted from enzyme induction since the duration of the experiments was always less than 90 min and the added substrate concentration was less than O-I pg glucose l-1. The uptake of glucose by water samples gave acceptable kinetics during the winter months with values for V,,, ranging between 0.~5 and 0.54 ng C l-1 h-l. During the summer, although the uptake rates were greater, they did not obey Michaelis-Menten kinetics. Consistently, less than 30% of the assimilated glucose was released as CO, by either water column or sediment microbes. Therefore, in the absence of data for the natural concentration of glucose, these data carmot be used to calculate the turnover of glucose in the estuary. Figure

192

I. R. Joint

Discussion The annual primary production of 143 g C rnw2 for the surface of the Lynher mudflat compares with values of IOO g C rnw2 for the Western Wadden Sea (CadCe & Hegeman, 1974), 81 g C mm2 for Southern New England (Marshall et al., 1971), zoo g C mm2 for a salt marsh in Georgia (Pomeroy, 1959) and 31 g C mm2 for the Ythan Estuary, Scotland (Leach, 1970). The mudflat of the Lynher has therefore a relatively high rate of primary production; indeed, the instantaneous rate of production must be greater than some other regions where photosynthesis continues when the mudflat is submerged. For example, Leach (1970) measured the same rate of photosynthesis for flooded and exposed sediments on the Ythan and could detect little vertical migration of diatoms related to the advancing tide. Vertical migration was not measured on the Lynher, but the inability to detect photosynthesis on submerged sediments suggests that a vertical migration of the microflora related to tidal coverage does, in fact, occur. As with many previous studies (Steele & Baird, 1968; Meadows & Anderson, 1968; Leach, 1970; Steele et al., 1970) considerable amounts of functional chlorophyll a and phaeopigments were found at depths well below the sediment photic zone. The mechanism of burial of this chlorophyll a is not known; it appears unlikely that diatoms would migrate such large vertical distances, although Harper (1976) states that in laboratory studies the freshwater diatom Pinnularia viridis is capable of migrating 3 cm h-l through sediment. None of the dominant species of deposit feeding benthos of the Lynher mudflat could be capable of transporting large concentrations of microflora to depths greater than IO cm (R. M. Warwick, personal communication). The burial of chlorophyll a was also unlikely to have been due to turbulence because the estuary was sheltered and the mudflat was stable, although drainage of pore water to the channels at low tide through the sediment may have resulted in the burial of microflora. However, the mechanism of transport of functional chlorophyll a to depths of 16 cm in the sediment is at present unknown; the phaeopigment concentration at depth was always greater than chlorophyll a, indicating that a degradation of the buried chlorophyll a was taking place, and that this layer of chlorophyll was nonfunctional in mudflat production processes. If a carbon to chlorophyll a ratio of 50 is assumed, microflora comprised 11% of the total carbon of the surface 0.5 cm sediment in the summer, 4% in March and 8% in November. Leach (1970) with a lower annual production, found the microflora standing crop of the Ythan mudflat was equivalent to 15% of the total carbon in the summer and 10% in the winter. The Enteromorpha bloom of 1973 in the Lynher did not appear to contribute chlorophyll a to the sediment, although the carbon content of the sediment at the beginning of the year was elevated. Harrison & Mann (1975) showed that, on death, the soluble components of the cells of Zostera were rapidly leached out, leaving only structural carbon which was degraded more slowly; the rate of leaching was independent of microbial activity. Since Enteromorpha is structurally more fragile than Zostera, it is likely that nitrogen and chlorophyll a would leach out of the dead Enteromorpha even faster leaving only structural carbon which increased the carbon content of the sediment at the beginning of this study. If the 1973 Enteromorpha bloom was as great as that in 1975, the maximum biomass would have been about IOO g C mp2, equivalent to an additional 40 mg C g-l dry weight of surface sediment: the total microflora production for 1974 was calculated to be 143 g C rnv2. Clearly, Enteromorpha when it occurs, must contribute significant amounts of carbon to the sediment. At present there is no satisfactory explanation for the increased nitrogen content of the sediment measured in June and July since this does not correspond with the maximum

Microbial production of an estuarine mudflat

193

biomass of the microflora or microfauna. The nitrogen maximum occurred at the same time as the maximum heterotroph biomass but was several orders of magnitude greater than could be accounted for by the nitrogen content of heterotrophic microbes. The numbers of heterotrophs in the surface sediment varied from 3 x 10~ g-l dry sediment in March to 2 x 10~ g-l dry sediment in August. Dale (1974) suggested that bacteria are present at depths greater than IO cm in intertidal sediments and he calculated a total heterotroph biomass of 9.5 g C mm2 in a fine silt sediment off Nova Scotia. Since no separate estimates of anaerobic microbes were made in this study, it was not possible to calculate the total biomass of heterotrophic microbes for the Lynher mudflat. Using the same conversion factors as Dale (1974), the numbers of aerobic microbes in the surface 0.5 cm sediment were equivalent to 0.6 mg C me2 in March and 400 mg C me2 in August: the maximum biomass of algae was 12 g C mV2. This study has provided few data on the rate of heterotrophic production: the uptake of [14C]-glucose was not a successful measure of the heterotrophic potential of the mudflat microbes. The deviation from Michaelis-Menten kinetics suggested that the microbial population of this mudflat did not have similar uptake kinetics, which is the basic assumption of this technique. The negative slopes of the modified Lineweaver-Burk plots obtained in the summer suggested enzyme synthesis, but this interpretation is unlikely to be correct because the incubation period of each experiment was short and the concentrations of added substrate were less than the natural substrate concentrations reported for other areas. The values of V,,, obtained for both the mudflat and the water column were comparable with those reported for the water column of the North West Atlantic, the tropical Pacific and an oligotrophic lake in Lapland (Crawford, Hobbie & Webb, 1973). On the basis of these measurements, there does not appear to be a rapid microbial transformation of organic carbon. If the input of carbon to the Lynher mudflat was considered to be largely autochthonous, would the measured primary production be sufficient to maintain the macrofauna production measured by Warwick & Price ? The total macrofauna production in 1973 was 13.3 g ash free dry weight, equivalent to 46 g C mV2 yr- l. Of the six major macrobenthic species present in the mudflat, Scrobicularia, Macoma and Ampharete are deposit feeders; assuming an ecological efficiency of IO%, they would have consumed 10.9 g C rnT2 during 1973. Two important species, Mya and Cerastoderma,are filter feeders, which would have required IO g C from the water column and from resuspended surface sediments. The nutrition of the major producer, Nephtys, is in doubt although Warwick & Price (1975) strongly suggested that it is a carnivore. Again, assuming a 10% ecological efficiency for each trophic level, Nephtys would have required a total primary production of 250 g C rnp2 yr-l. These calculations suggest a total annual requirement of the benthic macrofauna of 260 g C rnB2 from the sediment and IO g C rnb2 from the water column, but the calculated primary production of the sediment for 1974 was only 143 g C mm2 and that of the water column 82 g C ms2. Clearly the primary production of the sediment was insufficient to support the Nephtys population if the assumptions of ecological efficiency and carnivorous feeding are correct. The additional production of Enteromorpha may be sufficient to remedy this deficit, although it would be surprising if the survival of Nephtys depended on an occasional bloom of Enteromorpha. Heterotrophic microbes, although they utilize carbon, should probably not be considered as consumers where the source of carbon is autochthonous, since this study, and Williams (1970)~ have shown that less than 30% of the carbon taken up by such microbes is respired. Therefore, the efficiency of conversion of algal carbon into bacterial biomass is high: there

194

I. R. Joint

is little loss of carbon by respiration and the heterotroph biomass may be considered together with algal biomass as a food source for animals. However, if there was an important allochthonous source of carbon to the mudflat, heterotrophic activity would have to be much greater; although only one measurement of heterotrophic potential was statistically acceptable, the heterotrophic potential was apparently of the same order as not-very-productive oceanic waters. The rates of uptake of glucose by the samples which did not obey Michaelis-Menten kinetics were not significantly different from those samples which had acceptable kinetics, and in no experiment was there a very rapid uptake of glucose by the benthic heterotrophs, so it seems unlikely that there is a significant turnover of carbon by aerobic heterotrophs. An allochthonous source of carbon would have to be directly available to the mudflat fauna without microbial transformation; in terrestrial soils, from 50 to 70% of the plant litter is utilized by grazing herbivores and of the remaining litter 20% is utilized by bacteria and 80% by fungi (Gray, 1976). However, few fungi could be isolated on selective mycological media from the Lynher mudflat and it is doubtful if deposit feeders could directly utilize any plant litter transported downriver. Although attempts to measure the sedimentation of carbon to this mudflat in 1974 were unsuccessful, the low heterotrophic potential suggests that there was no significant allochthonous source of carbon and that primary production of the microflora of the sediment surface was the most important input to the sediment. Acknowledgements I wish to thank P. J. Radford for his guidance in statistics and A. J. Pomroy for technical assistance. This work forms part of the estuarine programme of the Natural Environment Research Council. References Cad&e, G. C. & Hegeman, J. 1974 Primary production of the benthic microflora living on tidal flats in the Dutch Wadden Sea. NetherlandsJournal of Sea Research 8, z6o-zgr. Crawford, C. C., Hobbie, J. E. & Webb, K. L. 1973 Utilization of dissolved organic compounds by micro-organisms in an estuary. In Estuarine Microbial Ecology (Stevenson, L. H. & Colwell, R. R., eds). pp. 169-177. Dale, N. G. 1974 Bacteria in intertidal sediments: factors relating to their distribution. Limnology and Oceanography 19, jog-518 Gray, T. R. G. 1976 Survival of vegetative microbes in soil. In The Survival of Vegetative Microbes. 26th Symposium of the Society for General Microbiology (Gray, T. R. G. & Postgate, J. R., eds). PP. 327-364. Harper, M. A. 1976 Migration rhythm of the benthic diatom Pinnularia viridis on Pond silt. New Zealand ‘journal of Marine and Freshwater Research IQ, 381384. Harrison, P. G. & Mann, K. H. 1975 Detritus formation from eelgrass (Zostera marina L.): relative effects of fragmentation, leaching and decay. Limnology and Oceanography ~0,gz4-g34. Leach, J. H. rg7o Epibenthic algal production in an intertidal mudflat. Limnology and Oceanography 15, 514-521. Marshall, N., Oviatt, C. A. & Skauen, D. M. rg7r Productivity of the benthic microflora of shoal estuarine environments in Southern New England. Internationale Revue der gesamten Hydrobiologie u Hydrographie 56, 947-956. Marshall, N., Skauen, D. M., Lampe, H. C. & Oviatt, C. A. 1973 Primary production of benthic microflora. In A Guide to the Measurement of Marine Primary Production Under Some Special Conditions. UNESCO, Paris. pp. 37-44. Meadows, P. S. & Anderson, J. G. 1968 Micro-organisms attached to marine sand grains. Journal of the Marine Biological Association U.K. 48, 161-175. Pomeroy, L. R. rg5g Algal productivity in salt marshes of Georgia. Limnology -and Oceanography 4, 386-m.

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Steele, J. II. & Baird, I. H. 1968 Production ecology of a sandy beach. Limnology and Oceanography 13, 14-q. Steele, J. H., Munro, A. L. S. & Giese, G. S. 1970 Environmental factors controlling the epipsammic flora on beach and sublittoral sands.Journal of the Marine Biological Association U.K. 50,907-918. Strickland, J. D. H. & Parsons, T. R. 1968 A manual of seawater analysis. BuEZetin of the Fisheries Research Board of Canada 125, 1-203. Warwick, R. M. & Price, R. 1975 Macrofauna production in an estuarine mudflat.rournal of the Marine Biological Association U.K. 55, I-IS. Williams, P. J. Le B. 1970 Heterotrophic utilization of dissolved organic compounds in the sea. I. Size distribution of population and relationship between respiration and incorporation of growth substrates. Journal of the Marine Biological Association U.K. 50, 859-870. Zobell, C. 1941 Studies on Marine Bacteria. I. The cultural requirements of heterotrophic aerobes. journal of Marine Research 4, 42-75.