Effects of destratification on autotrophic and heterotrophic microplankton productivity in eutrophic aquaculture ponds

Aquacukure, 50 (1985) 141-151 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands

EFFECTS OF DESTRATIFICATION ON AUTOTROPHIC HETEROTROPHIC MICROPLANKTON PRODUCTIVITY EUTROPHIC AQUACULTURE PONDS’

BARRY

A. COSTA-PIERCE

AND IN

and EDWARD A. LAWS

Department of Oceanography and Hawaii Institute Hawaii, Honolulu, HI 96822 (U.S.A.)

’ Hawaii Institute of Marine Biology Contribution (Accepted

141

of Marine Biology,

University of

No. 713

2 August 1985)

ABSTRACT Costa-Pierce, B.A. and Laws, E.A., 1985. Effects of de&ratification on autotrophic and heterotrophic microplankton productivity in eutrophic aquaculture ponds. Aquaculture, 50: 141-151. The effect of vertical mixing on microbial production rates in the water column of freshwater prawn (Macrobrachium rosenbergii) aquaculture ponds in Hawaii was examined using data collected over a period of 17 months. Ponds were judged as being stratified or well mixed based on the temperature difference between the top and bottom (depth 1 m). During stratified and well-mixed conditions this difference averaged 1.9 ? 0.8”C and 0.1 * 0.2”C, respectively. The seston of stratified ponds was found to be characterized by a significantly higher carbon/nitrogen (C/N) ratio (8.2 * 1.2 versus 4.0 f 0.6 by weight) and a significantly lower carbon/chlorophyll a (C/chl a) ratio (27 * 4 versus 80 * 55 by weight) than the seston of well-mixed ponds. However, there was no significant difference between stratified and well-mixed ponds in terms of mean chl (I concentration, or surface primary production rates and productivity indices. Vertical profiles of microbial DNA and RNA synthesis over a 40-h period during typical stratified and well-mixed conditions revealed a pronounced vertical gradient in the lower 0.5 m of the water column during stratified conditions, but only a 20-30% difference between stratified and well-mixed conditions in water-columnintegrated microbial DNA and RNA synthesis rates, and no significant difference in microbial growth rates as inferred from DNA/RNA synthesis rate ratios. Stratification events in these ponds are evidently of such short duration that microbial production and growth rates are rarely limited by the availability of organic and inorganic substrates.

INTRODUCTION

It is well known that the bottom waters of eutrophic ecosystems contain high concentrations of nutrients and that vertical mixing by wind turbulence, thermal convection and the bioturbation activities of fish and invertebrate populations contribute to, and in some cases control, the productivity

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o 1985 Elsevier Science Publishers B.V.

142

of these systems. Sediment resuspension and concomitant nutrient regeneration are prominent features of many aquatic environments, particularly shallow estuaries (Schubel, 1968) and fish ponds (Hepher, 1958). Indeed, one major constraint on the depth of fish ponds is the desire to allow nutrients regenerated at the sediment-water interface to be mixed into the euphotic zone and thus reduce the need for applied fertilizers (Maciolek, 1954). In shallow hypereutrophic fish ponds, the synthesis of organic matter and the addition of large amounts of supplemental feeds add more organic matter than can be decomposed. Organic matter rapidly accumulates on the pond bottom and is immobilized by burial, and this often leads to the development of anoxic conditions, production of toxic metabolities, and death of cultured organisms (Avnimelech et al., 1981). Little is known, however, of the effects of destratification and wind-driven resuspension on microbial activity and the distribution of autotropic and heterotrophic productivity. In this study we compared some selected chemical and microbial parameters measured under well-mixed and stratified conditions in commercial prawn (Macrobrachium rosenbergii) ponds to determine the effect of mixing on the chemical and microbiological characteristics of the water column of these systems. MATERIALS

AND METHODS

All experiments were conducted in freshwater 0.4-ha commercial prawn ponds, 1 m in depth, at Kahuku Prawn Co., Inc., Kahuku, Hawaii (KPC). Ponds were stocked, fed, and managed in accordance with conventional methods described by Malecha (1983). The ponds were sampled on a total of 10 dates from 13 February 1981 to 7 July 1982. The temperature at the pond surface and bottom was measured with a thermometer calibrated at 0.5% intervals. Subsurface water was collected by hand from the middle of the ponds using an alcohol-scrubbed, two-liter modified Meyer’s bottle (Lind, 1974). For primary production measurements, 150-ml aliquots were dispensed from this bottle into one light and one dark teflon-coated incubation bottle, the bottles spiked with 14C bicarbonate and incubated at the pond surface for l-3 h (11.00-14.00). A subsample from the same modified Meyer’s bottle was analyzed for inorganic carbon concentration using the pH titration procedure outlined in Strickland and Parsons (1972) with appropriate modifications for freshwater. At the end of the incubation period, aliquots from both the light and dark bottles were filtered onto Whatman GF/F filters. The filters were placed in liquid scintillation vials and frozen for transport to the laboratory. Upon return to the laboratory 1 ml of 1 N HCI was added to each vial and the acid bubbled overnight to remove residual inorganic carbon. The radioactivity in the vials was determined using a liquid scintillation counter with Aquasol as the fluor. Dark-bottle counts were subtracted from light-bottle counts to correct

143

for dark uptake of CO?. For particulate carbon and nitrogen analyses, quadruplicate 10 ml aliquots from the modified Meyer’s bottle were filtered through Whatman GF/F filters. The filters were placed on precombusted aluminum foil in clean Millipore petri dishes and immediately frozen for transport to the laboratory. The filters were subsequently analyzed for particulate carbon (PC) and particulate nitrogen (PN) on a Hewlett-Packard model 185B CHN analyzer following procedures recommended by Sharp (1974). Chlorophyll a (chl a) was measured on duplicate aliquots of 5-20 ml from the modified Meyer’s bottle. The samples were filtered onto Whatman GF/F filters and placed in darkened scintillation vials with 3 ml of absolute methanol. The vials were immediately put on ice and were stored at -20°C upon return to the laboratory. Chl a concentrations were measured fluorometrically using the procedures of Holm-Hansen and Riemann (1978) and Riemann (1980). Productivity indices (PI’s) were calculated by dividing the hourly rates of 14C autotrophic production by the chl a concentrations. On two dates, one during well-mixed conditions (18 September 1981) and the other during stratified conditions (8 October 1981), a water column profile of total protein was measured calorimetrically. Samples were collected at 20-cm depth intervals from the surface to 100 cm in an alcoholscrubbed Meyer’s bottle and quadruplicate g-ml subsamples were mixed with 1 ml of 6.1 molal trichloroacetic acid (TCA) in test tubes. The contents of the tubes were mixed thoroughly, and the tubes were capped and put on ice for transport to the laboratory. Upon return the samples were centrifuged at high speed for 45 min, the supernatant fluid aspirated off, and the remaining pellet was dissolved in 0.1 N NaOH to yield 0.1-0.3 mg protein*ml-‘. Aliquots of this mixture were neutralized with 0.15 N HCl, and the protein determined by a minor modification of the Coomassie Brilliant Blue dye-binding technique of Bradford (1976). During the summer of 1981 two 48-h experiments were conducted during well-mixed conditions (24-26 July 1981) and stratified conditions (14-16 July 1981) to determine microbial DNA and RNA production rates. On 14 July 1981 surface water was collected in a modified 2-l Meyer’s bottle and a 500-ml subsample added to a darkened polyethylene 2-l carboy at 07.30 h. At this time, and at 2-4-h intervals for 48 hours, triplicate lo-ml subsamples were removed from the carboy in the dark and 0.01 ml of 0.1 mCi*ml- ’ 3H-adenine stock solution added. After incubation in the dark for 0.5-h 2-ml subsamples were filtered through Whatman GF/F filters and frozen. On 24 July 1981 subsurface water was collected as above and incubated in a darkened polyethylene carboy with 3H-adenine beginning at 09.30 h as described above. At 2-6-h intervals for approximately 48 hours, triplicate lo-ml subsamples were taken from the carboy and processed as above. Relative DNA/RNA synthesis rate ratios were determined directly from the incorporation data according to the methods of Karl and co-workers (Karl et al., 1981a,b; Karl, 1981).

June July July June

81 81 81 82

in calculating

1.9+0.8

1.5 1.0 2.5 2.5

aNot included

Mean k SD

2 7 14 6

Mean fr SD 0.1+0.2

0 0 0 0 0.5 0

13 Feb.81 6 Apr.81 13Aug.81 8 Sept.81 29 June 82 7 July 82

of water column

Temp. diff. (“C, top-bottom)

1

Date

Summary

TABLE

the average.

2.1kO.9

1.13 2.73 2.93 1.62

Stratified

0.25kO.09

0.169 0.346 0.308 0.190

ponds

3.4* 1.8

0.16+0.15

0.379 0.109 0.116 0.051

0.22*0.12

8.2t1.2

6.7 7.9 9.5 8.5

4.OkO.6

4.6 4.4 4.3 3.5 4.2 3.1

0.217 0.237 0.343 0.354 0.094 0.060

1.99 3.68 2.49 6.78 1.87 3.58

9.16 16.2 10.7 23.7 7.84 11.1

1326

yz?)

~~g~ii’)

and 1982

Fzg*i-‘)

ponds

results on 10 dates in 1981

Well-mixed PC (mg*l“)

sampling

27*4

2.98a 25.0 25.3 31.8

80+55

42.2 68.4 31.2 66.9 83.8 185

PC/chl (g-g“)

a

0.17?0.08

0.225 0.109 0.260 0.093

0.31*0.20

0.276 0.332 0.446 0.603 0.105 0.067

Surface photosynthetic rate (mg C*l-‘*h-l)

1.4io.7

0.6 1.0 2.2 1.8

1.3tO.2

1.3 1.4 1.3 1.7 1.1 1.1

PI (g C-g-’

chl a-h-‘)

145

RESULTS

The ponds were classified as being well mixed or stratified based on the temperature difference between the top and bottom of the water column. If the temperature difference was 0.5% or less, the ponds were considered to be well mixed. If the temperature difference was 1.0% or more, the ponds were considered to be stratified. Since in reality there is a continuum of conditions ranging from truly well mixed through various stages of stratification, this classification scheme must obviously be viewed as an arbitrary one. However, in the absence of a much more extensive dat.a base, a more refined classification scheme seems unwarrented. The results summarized in Table 1 indicate that even this crude classification scheme revealed striking differences between the ponds. Based on a nonparametric Mann-Whitney U-test (Sokal and Rohlf, 1969) the PC and PN concentrations were significantly higher (P < 0.01) in the well-mixed ponds than in the stratified ponds. Furthermore, PC/PN and PC/chl a ratios were significantly lower and higher, respectively, in well-mixed ponds than in stratified ponds (P < 0.01). However, there was no significant difference between the well-mixed and stratified ponds in terms of chl a, surface photosynthetic rates, or productivity indices. The results of the water column profile work during well-mixed and stratified conditions were qualitatively in agreement with expectations. During well-mixed conditions there was no significant correlation between protein concentration or nucleic acid synthesis rates and depth (Figs. l-3). TOTAL

Fig. 1. ditions Points dition.

PROTEIN

CONCENTRATION

(pg.f-‘)

Depth profile of total chemical protein concentrations during well-mixed con(X ) on 8 September 1981 and stratified conditions (0) on 8 October 1981. are mean values of quadruplicate samples taken from two ponds of each conStandard deviations were calculated using the eight samples from the two ponds.

146 TOTAL MICROBIAL PRODUCTIVITY

BY RNA SYNTFIESIS

(,gRNA

I-’ h-')

Fig. 2. Depth profile of total RNA synthesis rates. Symbols and points as in Fig. 1.

TOTAL

0~ii---

MICROBIAL

2.5

5.0

PRODUCTIVITY

7.5 1

10.0 I

BY DNA

12.5 I

SYNTHESIS 15.0 I

( pgDNA,lw’,h

17.5 1

20.0 I

-‘)

245

20 -

2 40“;:

,

x

1 ii g 60 -

Fig. 3. Depth profile of total DNA synthesis rates during well-mixed conditions 14 ApriI 1982 and stratified conditions (0) on 6 June 1982. Points as in Fig. 1.

(X )

on

However, during stratified conditions there was a highly si~~ic~t prositive correlation fr = 0.93, P < 0.01) between protein concentration and depth throughout the water column (Fig. 1). Significant positive correlations were also observed between DNA synthesis rates and depth (r = 0.99, P < 0.01)and between RNA synthesis rates and depth (r= 0.95,P < 0.05), but these latter correlations were confined to the lower 60 cm of the water column. Water-column-averaged protein concentrations were about twice as high under well-mixed than under stratified conditions (1.2 versus 0.6 mg*l-’ protein), but there was only about a 22% difference in DNA synthesis rates (12.2 versus 9.8 pg DNA* l-r* h-’ for well-mixed and stratified conditions, respectively) and a 30% difference in RNA synthesis rates

147

(140 versus 104 pg RNA-l-‘oh-’ for well-mixed and stratified conditions, respectively). Based on a t-test, there was no significant difference (P > 0.10) in mean DNA/RNA synthesis rate ratios during well-mixed (0.080 it 0.023) and stratified (0.087 + 0.007) conditions (Fig. 4).

.05 0800

1200

1600

2000

2400

04.00

LOCAL

I 08.W

\x

1290

16.00

2C&30

I 24.00

TIME

Fig. 4. Temporal variation in dark DNA/RNA synthesis rate ratios course during stratified (14-16 June 1981) and well-mixed (24-26 ditions. Symbols as in Fig. 1. Points are means of triplicate samples

over a 40-h time July 1981) confor each interval.

DISCUSSION

That mixing greatly altered the concentration of the particulate carbon and nitrogen in the water column is obvious from an examination of the data in Table 1. This increase in PC and PN undoubtedly resulted in large part from a resuspension of bottom sediments. This result in itself is hardly surprising, but a closer examination of the data leads to some unexpected conclusions. A comparison of PC/PN ratios during well-mixed and stratified conditions (Table 1) shows that the seston was much richer in nitrogen during well-mixed conditions. In fact, the PC/PN ratio of the seston during well-mixed conditions (4.0 + 0.6) is comparable to that of pure protein (3.3 by weight) (DiTullio and Laws, 1983). However, an examination of Fig. 1 indicates that protein itself probably accounted for very little of the increase in PC and PN. The water-column-averaged protein concentration during well-mixed conditions was only 1.2 mg-l-‘, which corresponds to protein carbon and nitrogen concentrations of about 0.6 and 0.18 mg*l-’ respectively (Jukes et al., 1975; Holmquist, 1978). These figures are less than 5% of the average PC and PN concentrations measured in the well-mixed ponds. A similar calculation based on the surface-water

148

protein concentration measured during stratified conditions yields protein carbon and nitrogen concentrations equal to 10% and 26’S, respectively, of the average PC and PN concentrations measured during stratified conditions. Thus, despite the low PC/PN ratio of the seston during well-mixed conditions, protein appears to have accounted for a higher percentage of the seston during stratified conditions. An additional clue to the composition of the seston comes from an examination of PC/chl a ratios. In the stratified ponds this ratio averaged 27 + 4 by weight, a figure close to the lower limit of PC/chl a ratios observed in microalgae (Laws and Bannister, 1980). Thus, the particulate material near the surface of the stratified ponds must have consisted almost entirely of phytoplankton, since a substantial contribution by non-phytoplankton particles to this material would necessarily imply that the PC/chl a ratio of the remaining phytoplankton particles was substantially less than 27, a very unlikely condition judging from the available experimental data base on phytoplankton composition (Laws et al., 1983). It therefore seems reasonable to conclude that the mean PC/PN ratio of 8.2 measured during stratified conditions is probably close to the PC/PN ratio of the phytoplankton cells in these ponds. The relatively low PC/chl a and PC/PN ratios of the phytoplankton, as well as the low light-saturated productivity indices (1.3-1.4), are all indicative of phytoplankton growing in a strongly lightlimited system (Laws and Bannister, 1980; Laws et al., 1985). When these PC/chl a and PC/PN ratios are used to interpret the data from the wellmixed ponds, the estimated concentration of phytoplankton carbon is found to be (0.22 mg chl a-l-‘) (27) = 5.9 mg PC*l-‘, and the concentration of phytoplankton nitrogen is estimated to be (5.9 mg PC*l-‘)/(8.2) = 0.72 mg PN*I-‘. Thus, during well-mixed conditions, phytoplankton is estimated to account for (100%) (5.9/13) = 45% of the PC and only (100%) (0.72/3.4) = 21% of the PN in the water column. The PC/PN ratio of the non-phytoplankton seston in the well-mixed ponds is estimated to be (13 - 5.9)/(3.4 - 0.72) = 2.6 by weight. From the foregoing analyses we conclude that the particulate material resuspended into the water column during well-mixed conditions must have consisted in large part of non-proteinaceous but very nitrogen-rich particles. These particles may have consisted in part of nitrogen-rich organic compounds (e.g. urea has a C : N ratio of 0.43 by weight), ammonium adsorbed to clays, and complexes of inorganic clays and nitrogen-rich detritus. Decaying marine plankton has been shown to release nitrogenrich organic compounds with a C : N ratio less than 3.0 in an experiment conducted in the absence of zooplankton (Otsuki and Hanya, 1968). Odum et al. (1979) have found that as much as 30% of the nitrogen content of detritus in freshwater lakes and streams may exist in the form of nonprotein nitrogen compounds and that the nitrogen increase which often accompanies initial stages of vascular plant decomposition cannot be attributed solely to an increase in microbial protein.

Boyd (1979) has noted that nitrogen often limits the rate of decomposition of organic matter in aquatic systems, and that organic matter with a low C : N ratio therefore tends to decompose more rapidly than organic matter with a high C : N ratio. We had therefore expected microbial nucleic acid synthesis rates to be subst~ti~ly higher during wellmixed conditions, when PC/PN ratios were approximately half those measured under stmtified conditions. However, a direct comparison of nucleic acid synthesis rates revealed only a 20-30% increase under well-mixed conditions, DNA/RNA synthesis rate ratios have been shown to vary in direct proportion to the specific growth rate of m~~roorganisms~and may be used for estimating and comparing the productivities of microbial assemblages in nature (Karl, 1980, 1981). The fact that the DNA/RNA synthesis rate ratios were so similar under well-mixed and stratified conditions (Fig. 4) supports our contention that resuspension of bottom sediments had little effect on the activities of the microorganisms in the water column What explanations can be offered for this lack of response? First, ~guments concerning the ease of decomposition of organic matter are pertinent to bacterial, but not phy~p~ankton, production. Although our dark incubations undoubtedly discriminated against phytopl~kto~ growth, phytopia~kton is capable of synthesizing RNA and DNA in the dark. To what extent did phyt~pl~kton production contribute to the observed rates of nucleic acid synthesis? An upper bound on the contribution of phytoplankton to the observed DNA synthesis rates may be calculated by noting that the DNA/PC ratio in microbic cells ranges between 1 and 3% by weight (Karl and Winn, 1984). Since photosyn~etic rates measured at the pond surface undoubtedly constitute an upper bound on water-~ol~rn~-averted photosynthetic rates, upper bounds on the contribution of the phytopl~kt~~ to DNA synthesis in the dark are (0.31 mg C*l-‘-h-l) (0.03) = 9.3 g DNA*l-‘*h-’ and f0.17 mg C+l-“-h-l) (0.03) = 5.1 g DNA*]-“*h-l in the well-mixed and stratified ponds, respectively. These figures are 50-75% of the average microbial DNA synthesis rates which we measured. Thus, while it is possible that a non-trivia amount of the nucleic acid synthesis we measured was due to the activities of phytoplankton, the majority of the activity was most likely due to bacteria. The failure of the bacterial ~orn~~nity to respond in a more dramatic way to the resuspension of bottom sediments suggests either that bacteriaX production was not substrate limited or that some critical substrate rern~~~ limiting even in the presence of resuspended sediments. Our data allow no clear choice between these two alte~ative explanations, but the high rate of applied feed inputs to these ponds (40 kg*ha”‘*d-’ dry weight) would tend to favor lack of substrate limitation as the probable explanation. Our data indicate that, in addition to having fittle apparent effect on bacterial probation rates, vertical mixing had no s~n~ic~t stimulator effect on phyt~pl~kton productive. Based on the observed light-~turat~d productivity indices and the estimated PC/PN and PCfehl a ratios of the

150

phytoplankton, this result is not particularly surprising. The relatively low values of all these parameters suggest that the phytopl~kton was light rather than nutrient limited (Laws and Bannister, 1980). Since the resuspension of bottom sediments undoubtedly reduced the transmission of light through the water column, vertical mixing may actually have decreased rather than increased water column primary production rates. The failure of the microorganisms in these ponds to respond to the resuspension of bottom sediments does not necessarily imply that vertical mixing is unimportant in the ecology of these ponds. On the contrary, the shallow depth of the ponds and- the persistent nature of the northeast Trade Winds cause well-mixed conditions to be the rule rather than the exception in these systems. Data collected by the Hawaii Natural Energy Institute indicate that in this area the Trade Winds blow at speeds of ‘7 2 3 m/s (16, + 7 mph} at an elevation of 9 m (HNEI, 1983). Thus windy days are the rule rather than the exception in this area. This information suggests that periods of stratification are short-lived phenomena in these ponds, and our results indicate that the internal nutrient reserves of the microorganisms and the concentrations of dissolved organic and inorganic nutrients in the water are sufficient to sustain the growth of the bacteria and phytoplankton during these stratification events.

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151 Karl, D.M. and Winn, C.D., 1984. Adenine metabolism and nucleic acid synthesis: applications to microbiological oceanography. In: J.E. Hobbie and P.J. LeB. Williams (Editors), Heterotrophic Activity in the Sea. Plenum, New York, pp. 197-215. Karl, D.M., Winn, C.D. and Wong, D.C.L., 1981a. RNA synthesis as a measure of microbial growth in aquatic environments. I. Evaluation, verification, and optimization of methods. Mar. Biol., 64: l-12. Karl, D.M., Winn, C.D. and Wong, C.D.L., 1981b. RNA synthesis as a measure of microbial growth in aquatic environments. II. Field applications. Mar. Biol., 64: 13-21. Laws, E.A. and Bannister, T.T., 1980. Nutrient- and light-limited growth of Thalassiosira fluviatilis in continuous culture, with implications for phytoplankton growth in the oceans. Limnol. Oceanogr., 25 : 45 7-47 3. Laws, E.A., Karl, D.M., Redalje, D.G., Jurick, R.S. and Winn, C.D., 1983. Variability in ratios of phytoplankton carbon and RNA to ATP and chlorophyll a in batch and continuous cultures. J. Phycol., 19: 439-445. Laws, E.A., Jones, D.R., Terry, K.L. and Hirata, J.A., 1985. Modifications in recent models of phytoplankton growth: theoretical developments and experimental examination of predictions. J. Theoret. Biol., 114: 323-341. Lind, O.T., 1974. Common Methods in Limnology. C.V. Mosby, St. Louis, MO. Maciolek, A., 1954. Artificial fertilization of lakes and ponds. Spec. Sci. Ref., U.S. Fish Wildlife Service, No. 113,49 pp. Malecha, S.R., 1983. Commercial pond production of the freshwater prawn, Macrobrachium rosenbergii, in Hawaii. In: J.P. McVey (Editor) CRC Handbook of Aquaculture, Vol. 1, Crustacean Aquaculture. CRC Press, Boca Raton, FL, pp. 231259. Odum, W.E., Kirk, P.W. and Zieman, J.C., 1979. Non-protein nitrogen compounds associated with particles of vascular plant detritus. Oikos, 32: 363-367. Otsuki, A. and Hanya, T., 1968. On the production of dissolved nitrogen-rich organic matter. Limnol. Oceanogr., 13: 183-185. Riemann, B., 1980. A note on the use of methanol as an extraction solvent for chlorophyll a determination. Arch. Hydrobiol. Beih. Ergebn. Limnol., 14: 70-79. Schubel, J.R., 1968. Turbidity maximum of the Northern Chesapeake Bay. Science, 161: 1013-1015. Sharp, J.H., 1974. Improved analysis for “particulate” organic carbon and nitrogen from seawater. Limnol. Oceanogr., 19: 984-989. Sokal, R.R. and Rohlf, F.J., 1969. Biometry. W.H. Freeman and Co., San Francisco, CA, 776 pp. Strickland, J.D.H. and Parsons, T.R., 1972. A Practical Handbook of Seawater Analysis. Bull. 167, Fish. Res. Board Can., Ottawa, Ont., 310 pp.