Aquacultural Engineering6 (1987) 259-275

Automatic Control of Food Supply in the Culture of Filter-feeding Organisms

William. L. Huggins
Ministry of Agriculture, Fisheries and Food, Directorate of Fisheries Research, Fisheries Laboratory, Lowestoft, Suffolk NR33 OHT, UK

Michael M. Helm and Dennis R. Williams

Ministry of Agriculture, Fisheries and Food, Directorate of Fisheries Research, Fisheries Laboratory, Benarth Road, Conwy, Gwynedd LI_32 8UB, UK

ABSTRA CT The design, construction and operation of a laboratory apparatus is described, which opto-electronically controls food density within the optimum range necessary to promote the maximum growth of filter-feeding organisms. This apparatus, termed an algal cell monitor, incorporates an infra-red transmitter and photo-receiver with circuitry to permit stable operation over a wide range of culture conditions. Application of the apparatus in controlling the density of microalgal food species in the highdensity rearing of oyster larvae, together with cost benefits of the technique, are discussed.

INTRODUCTION
Of great importance in the culture of filter-feeding organisms is the way in which their food ration is given. Bivalve molluscs, which filter microscopic algae cells from the surrounding water, achieve the maximum rate of growth in conditions of continuous food supply, but with relatively narrow limits of food cell density. When the optimum food density is exceeded, some of the filtered cells are wasted, being voided as pseudofaeces, and the cells that are ingested may be digested inefficiently. In, for example, bivalve hatcheries where larvae and juveniles of oysters and clams are reared intensively, the efficient use of cultured algae, which are costly to produce, is very important (Helm and Laing, 1981 ). Maximum efficiency would be achieved by the automatic replen259

Crown Copyright. 1987

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W. L. Hug,gins, 3/1. M. Helm, D. R. Williams

ishment of food grazed by the animals so that the optimum food cell density is maintained at all times. The use of automatic devices to control nutrient input in the culture of microorganisms is described by Powell (1963) and Munson (1970). Winter (1973) described the use of a photometric, automatic feeding apparatus in critical determinations of filtration rate in the mussel, Mytilus edulis. This. device, described by Haupt (1979), utilised a lightdependent resistor to detect changes in the attenuation of a light beam caused by variation in the concentration of food cells suspended in the culture medium. The signal was compared with a reference voltage, which could be varied to alter the required food cell concentration. An electromagnetic valve was actuated when the concentration fell below that corresponding to the reference voltage and food was added to the experimental container to restore the required concentration. This report describes the design and construction of a similar apparatus, termed an algal cell monitor (ACM), developed independently at the MAFF Fisheries Laboratory, Lowestoft, specifically, but not exclusively for use in controlling food cell density in the intensive culture of larvae of the European flat oyster, Ostrea edulis, at the MAFF Fisheries Laboratory, Conwy. The purpose of this device was to evaluate the possibility of rearing larvae in conditions of controlled feeding at greater densities than previously had been practical with manual feeding methods. This was a move towards a more economical production of oyster juveniles (spat). Results are given of a comparative series of trials in which O. edulis larvae were reared at a density of 1.4 ml- ~with manual food addition (Walne, 1974) and at greater densities using the ACM. Information is provided on the feeding rate of larvae, their growth and survival to metamorphosis and the production of spat with the two culture methods. The cost benefits of high-density rearing are discussed. MATERIALS AND METHODS
T h e algal cell monitor -- design criteria

An apparatus was required that would fulfil the following specifications: 1. Control the cell concentration of algae in culture vessels within the optimum range to support the maximum growth rate of larvae. 2. Be capable of rapid calibration to discount the effects of density of larvae, the increase in size of larvae as they develop and changes in the species composition of the food ration. 3. The sensor should not be affected by changes in ambient illumination in the culture area.

Food supply in culture of filter-feeding organisms

261

4. The circuit should be temperature compensated to provide operational stability in cultures where minor variations in temperature can be expected. 5. The apparatus should be portable and capable of operation in a range of culture vessels and situations.

Operating principle
The ACM described here is fitted with an infra-red transmitter and photo-receiver. It is insensitive to ambient light and use of the infra-red source discourages settlement of algae on the transmitter window. Interference by substances that discolour the culture medium is reduced. In operation, the-receiver produces a current proportional to both the concentration of the algal cells used to feed the larvae and the opacity of the sea-water medium. If a suitable resistor is included in series with the receiving element, the voltage produced across it is proportional to the algal concentration. This voltage is compared with a reference voltage and if greater (indicating a fall in algae concentration) activates a peristaltic pump. Equilibrium is reached when a sufficient volume of algae is pumped to just replenish that grazed by the larvae. When the received voltage falls below the pre-set reference level the control circuit switches the pump off.

Design and construction of the sensing unit

A schematic diagram of the whole system is shown in Fig. 1 and some of the component parts in Fig. 2. The sensor unit was designed with watertight seals to permit immersion in rearing tanks containing oyster larvae (Figs 3 and 4). The sensor unit contains an infra-red transmitter, which is aligned with a suitable receiver over a path length of 250 mm (MHS 400 and MRH 400, respectively, manufactured by MCP Electronics Ltd, Middlesex, UK). The transmitter uses between 4 and 5 mW of power from the 20-V supply at the normal settings. Each unit consists of an active element mounted in a black, anodised, machined, aluminium block, with a 15.9 mm diameter biconvex lens t o produce a quasiparallel beam with a diameter of approximately 10 mm and a spread of approximately 1 in water. The units are mounted within a 133 mm diameter stainless steel bulkhead with a glass or acrylic window made watertight by an 'O' seal. Each unit is totally enclosed with the connect: ing lead sealed with epoxy resin (Two-tube Araldite). The material for the leads must be non-toxic: PVC and silicone rubber sheathed cables have proved suitable.

Control

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Sensor probe Larval rearing vessel

I= r r "

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so.roe housing

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Fig. I.

Monitor system arrangement.

Food supply in culture of filterTfeeding organisms

263

Fig. 2.

The sensor unit, control box and peristaltic pump.

Circuit details

The circuit diagram of the ACM is shown in Fig. 5. The infra-red transmitter is a gallium arsenide (GaAs) light-emitting diode (MCP Electronics Ltd)(MGA 5000), which emits fight in the near-infra-red region at a wavelength of approximately 900 nm. At this wavelength, silicon photo-detectors operate at their peak efficiency and most glasses or clear acrylics can be used in the optical path. The detector is an MSP6 composite silicon photo-receiver (MCP Electronics Ltd) with peak spectral response at 950 nm. This is used in series with a resistor and thermistor chain to provide the voltage signal (V~x), which is fed to the inverting input (pin 2) of the operational amplifier RC741C via a 22 kff2 resistor. Inclusion of the thermistor, fitted into the aluminium block holding the detector, provides stability of + 0.04% at the temperature used in the rearing of larvae (24C + IC). A 4.7-/~F capacitor between pin 2 and 0 V helps to smooth out circuit response due to the passage of air bubbles and fluctuating concentrations of larvae across the optical path. The reference voltage (Vree) derived from part of a resistor chain is applied to pin 3 of the 741 operational amplifier, its value being adjusted by the two potentiometers in the chain, giving 'coarse' and 'fine' control. This setting of Vref is adjusted to the optimum concentration of algae cells in suspension for the growth of the larvae (see calibration procedure described later). For a concentration of 100 cells /x1-1 of Isochrysis galbana, V~e~is set between 8.5-9.0 V. Exact determination depends on

264
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W. L. Huggins, M. M. Helm, D. R. Williams

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Food supply in culture of filter-feeding organisms

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Fig. 4.

The sensor unit.

the colour and clarity of the sea water used in rearing, and account is taken of these factors in the calibration procedure. A change in food cell concentration from 100 to 75 cells ~1 -~ gives a voltage change of approximately 2 V for V~x.Feedback is applied between the output (pin 6) of the operational amplifier and pin 3. The degree of hysteresis of the comparator can be controlled by choice of a suitable feedback resistor; in this case a 2M2 resistor gives a hysteresis range of 0.2 V about the reference voltage level. The upper hysteresis point is 0.07 V higher than Vref and the lower point O.13 V below the reference. Operation of the 741 circuit is such that if Vr~ is less than the lower hysteresis point the output is high, approaching the supply voltage. For a receiver voltage greater than the upper hysteresis point the output is low, approaching 0 V. When Vr~ is greater than the higher reference point, indicating a reduced algal cell concentration, the output of the 741 is low, holding the BC183LC transistor in an OFF state. In this state the relay is not energised and power is applied to the pump. With the pump in operation the receiver voltage falls as the algal cell concentration rises towards the optimum. When it falls below the lower hysteresis point, the output of the 741 increases and turns on the BC183LC, thus energising

266

W. L. Huggins, M. M. Helm, D. R. Williams

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Food supply in culture offilter-feeding organisms

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the relay, which switches off the power to the pump. Provision is made for a Rustrak recorder model 88 (Gulton Ltd, Middlesex, U K ) ( 0 - 1 0 mV) to monitor pumping activity.

Calibration procedure
1. The sensor unit is immersed in a larval rearing vessel containing freshly filtered sea water (Fig. 6), the larvae and a sufficient volume of the preferred algal species to raise the food cell concentration to the optimum level. A uniform suspension of food and larvae is achieved by aerating the culture. 2. The 'fine' potentiometer control is turned to its mid-point setting. 3. After allowing 2 min for the culture to equilibrate, the control unit is connected to the mains supply via the isolation transformer. The 'coarse' control is adjusted until a setting is found that will just light or extinguish the 'pump-on' indicator lamp; a finer setting can be obtained with the 'fine' control. 4. The cell monitor will now operate to maintain the required algae cell concentration in the rearing vessel provided the pump is able to provide a suitable concentrate of the algae to replenish those cells grazed by the larvae.

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Fig. 6.. Calibrating the system.

268

W. L. Huggins, M. M. Helm, D. R. Williams

The cell concentration decreases with time because of the increase in size of the larvae and the colouring and fouling of the water with metabolites and food debris. It is therefore necessary to recalibrate periodically to correct drift and maintain the required cell concentration. A decrease, amounting to 10-30% of the required cell concentration, does not impede the growth rate of the larvae.

Rearing methods
The benefits of controlling the availability of food with the ACM in the large-scale rearing of O. edulis larvae were investigated in five trials. In each, a batch of larvae was divided between two 125-1itre polyethylene rearing vessels (Walne and Helm, 1974). One vessel was run at a low density of larvae ( 1.44 ml- 1) as a control experiment, and the other at a density 5.0 to 8.6 times greater (7.3 ml- 1 to 12.4 ml- l). Vessels were filled with sea water at salinities of between 31 and 34%0 and at 24C. This was finely filtered to remove 90% of the naturally occurring particulate material greater than 2.5 ~tm diameter and pumped through an ultra-violet lamp unit to reduce its bacterial content (Wickins and Helm, 1981).

Low-density rearing
Control experiments at low larval density were operated according to the procedures used at this laboratory for many years (Walne, 1974). Sea water was changed each week on Mondays, Wednesdays and Fridays, and the larvae were fed once daily with a mixture of 33 cells pl-~ each of/. galbana and Chaetoceros calcitrans together with 3.3 cells pl-~ Tetraselmis suecica (Helm and Millican, 1977). At each water change the larvae were removed, washed and counted, and a sample of 100 was taken for shell length measurements. The survivors were returned to freshly filtered water in a clean vessel.

High-density rearing
Feeding of larvae reared at the higher densities was controlled by the ACM. Algae to be used in maintaining the optimum food cell density were stored in a 10 litre reservoir kept at 15 to 18C by cool fresh water (see Fig. 1 ). To provide food in the best possible condition the reservoir was cleaned and refilled daily with the required species in the correct proportions by cell density. Tubing leading from the reservoir to the peristaltic pump and from the pump to the rearing vessel was cleaned daily to minimise microbial contamination. High-density cultures received a daily water change. This was necessary to reduce the risk of mortalities resulting from severe fouling in the

Food supply in culture offilter-feeding organisms

269

form of algal debris, water discoloration due to metabolites and decomposition products and high levels of bacteria. At each water change the sensor of the ACM was removed from the vessel and thoroughly washed, with particular attention to the transmitter and receiver windows, to remove adhering algal debris and bacteria. Larvae were washed, counted and a sample measured at each change. Following water changes the sensor was replaced in the rearing vessel to which the larvae had been returned and the volumes of the algal species necessary to enrich the water, by 25 cells/zl-1 each of Isochrysis and Chaetoceros and 2.5 cells/zl-J Tetraselmis, were added. The use of lower food cell densities than in low-density larval rearing was a precautionary measure in the event of instability of the ACM. In practice, stability proved to be excellent in all trials. The even dispersion of food cells in the culture volume was maintained by aerating at about 100 litres h-t. After allowing 2 min for the mixing of the food the ACM was calibrated following the procedure described earlier. At the end of each 24-h period, estimates were made of the residual cell concentration of algae in the rearing vessels and of the volume and density of algal culture remaining in the supply reservoir. These enabled calculations to be made of the rate of ingestion of algal cells by larvae for comparison with rates calculated for low-density experiments. Cell counts were made using a Model ZB Coulter Counter.
Settlement of larvae

In four trials, cultures were continued through settlement so that yields of spat could also be compared. When larvae reared at high density reached the settlement phase, they were reduced to a maximum of 2 ml-~ by division between the appropriate number of 125-1itre vessels. A single, black, matt-surfaced, PVC disc was placed in each of the vessels and this was removed daily to detach the settled spat (Walne and Helm, 1974). Daily counts were made of the numbers of spat that settled in each treatment. During settlement, food was provided once daily as in low-density larval rearing. RESULTS AND DISCUSSION
Food cell clearance rates and larval growth

Data collected during the course of the trials enabled calculation to be made of the rate at which algal cells were cleared from suspension by larvae of different mean shell lengths (Fig. 7). Clearance rates were

270
140

W. L. Huggins, M. M. Hehn. D. R. Williams

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Fig. 7. The daily cell clearance rate (thousands of cells equivalent in size to lsochrysis cleared larva-1) related to mean shell length in high-density rearing ( ) and lowdensity control (---- --) cultures. Regression equations are provided in the text.

calculated as cells equivalent to Isochrysis grazed in 1 day where 1 cell of Tetraselmis was equivalent to 10 cells of either Isochrysis or Chaetoceros in terms of ash-free dry weight. Best-fit equations expressing the relationship between clearance rate and shell length for larvae reared at low and high densities were as follows: Low density: y= 591"63Z- 82077.28 (r= 0"806, 30 degrees of freedom)

(1)
High density: y= 906"07Z- 134882-16 (r= 0"812, 34 degrees of freedom)

(2)
where y= number of cells cleared per larva day-l, and Z = mean shell length of larvae (/~m). Larvae at high density with automatically-controlled food supply cleared considerably more cells per day than those at low density, with daily food addition (Fig. 7). This was particularly evident as they approached the size at which they settle. A larva of 280 ~m shell length at high density cleared 118 800 cells day-1 compared with 83 600 cells day- 1 for a larva of the same shell length at low density (a difference of 42%). For each 10-/~m increment in shell length a larva cleared an addi-

Food supply in culture of filter-feeding organisms

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271

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tional 9061 cells day -~ at high density and 5916 cells day -~ at low density. Despite the differences in clearance rate, growth in high-density rearing was similar to that at low density. Possible explanations for this are a greater rate of pseudo-faeces production and/or the inefficient digestion and absorption of food related to a reduced gut retention time in automatically fed larvae cultures. Growth data from high-density rearing trials are shown in Fig. 8 and the regression line is fitted from the exponential equation: logey= 0"070?( + log~ 171-480 ( r-- 0.971, 34 degrees of freedom)

(3)
The best-fit equation for the growth of larvae at low density was: logey= 0.068?( + log~175-054 (r= 0"968, 17 degrees of freedom) (4) where y = shell length of the larvae (/~m), and 2: -- time in days. Starting the trials at a shell length of about 170 ~m, larvae at high density reached 300 ~m by day 8 compared with a shell length attained at low density of 301/~m.

272

W. L. Huggins, M. M. Helm, D. R. Williams

Survival of larvae

Comparisons of survival during the pelagic veliger stage in both highdensity cultures and the low-density controls are shown in Table 1. Observations of the density of larvae prior to settlement (Dl) were made on day 8 or 9 when the majority of the survivors had reached the pediveliger stage with well-developed eye spots. In four of the five trials at the low density, overall survival until the pediveliger stage ranged from 72% to 96% while in the fifth it was 43%, following an unusually high mortality rate between days 3 and 5. In the high-density trials survival ranged between 48% and 69%. Losses of larvae in all cultures were accounted for partly by mortalities, but mainly by the grading out and rejection of slower growers. Survival at the high density was related to days of rearing from the time of liberation (day 0) by the equation: y= - 4"50X+ 95.97 (r= 0"791, 36 degrees of freedom) (5) where y is percentage survival of the initial number of larvae and Z is time in days (Fig. 9).

TABLE 1 Density of Larvae Stocked Initially (Do) and Surviving Immediately Prior to Settlement (D~) in Five Comparisons of High- and Low-density Rearing in 125-1itre Vessels

Trial

Treatment

Density of larvae (ml- i) Do DI

4.64 0"62 4.80 1.22 7.20 1"38 8.54 1.17 4.54 1.04 5"94 1"09

Survival (%)

Start of settlement

Spat yieM

(d)
1 2 3 4 5 Mean High density Low density High density Low density High density Low density High density Low density High density Low density High density Low density 9-76 1.44 7.31 1.44 12.00 1.44 12.38 1.44 8.31 1.44 9.95 1"44 47"54 43"06 65"66 84.72 60"00 95"83 68-98 81.25 54-63 72"22 59.70 75.42

(%)

11 10 10 9 9 10 10 10 10

24.6 43.3 43.8 39"5 52'0 38"6 41.6 40.7 23"9 40"5 ~ 40"3 a

a Means apply to Trials 2 to 5 inclusive.

Food supply in culture of filter-feeding organisms

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2 73

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The percentage survival of larvae grown at high density related to time in days from liberation.

TABLE 2
Daily Volume of Algae Needed to Satisfy the Nutritional Requirements of the Average Batch of 1.24 Million O. edulis Larvae in High-density Rearing a

Day

Mean larval length (/~m)

171 184 197 211 227 243 261 279 300 322

Number of larvae ( x 10 ~)
1.244 1.165 1"090 1"015 0.940 0"875 0"825 0'780 0"755 0.743

Cells ingested (per larva in 24 t7)

Vol. of algae~ ingested (litre- i)

2.49 3"71 4.75 5.71 6-66 7.46 8"38 9'20 10"34 11.65 Total 70.35

0 1 2 3 4 5 6 7 8 9

20 060 31 830 43610 56 300 70800 85290 101600 117910 136 940 156 870

"Algal volumes are in 1 at 10 000 cells pl-~ lsochrysis equivalents. hWastage (i.e. volume of algae uneaten and lost at water changes) averaged 0.625 litres day-~ and needs to be added to each day's total to give actual total volume provided, which was 76.0 litres until spatfail commenced.

274

W. L. Huggins, M. M. Helm, D. R. Williams

This relationship is used later to calculate, in Table 2, the numbers of larvae remaining at successive days of rearing for the average brood. Settlement of larvae Within a trial, larvae generally started to settle on the same day whether they were reared at high or low density. In three trials this was day 10 and in the other two either day 9 or 11 (Table 1 ). Larvae from both highand low-density cultures were pooled for settlement in Trial 1, but were settled separately in each of the other four. It will be noticed in Table 1 that, whereas survival to the pediveliger stage averaged 75-4% in the low-density cultures compared with 59.7% in high-density cultures, the average spat yields were 40.3% and 40.5% of the initial number of larvae, respectively. Evidently, a greater percentage of pediveligers in low-density cultures failed to settle. This is probably because of increased difficulty in grading out and discarding slow-growing and moribund larvae from the low-density systems where the size distribution within the population was relatively compact. In contrast, there was a much more widely spread distribution of sizes in high-density cultures. Cost benefits of high-density rearing A comparison of some factors contributing to the cost of rearing the average batch of 1.24 million O. edulis larvae through to settlement at
TABLE 3 Summary of Factors Contributing to the Cost of Rearing the Average Batch of 1.24 Million O. edulis Larvae Through to Settlement at High- and Low-larval Densities

Larval density

High
Number of 125-1itre rearing vessels Mean time to settlement (days) Number of water changes per vessel Total volume of filtered, heated, sterilised sea water required (litres) Total man hours to rear larvae to settlement Total volume of algae required to rear larvae to settlement (litres at 10 000 cells/~1-1) Number of spat produced 1 10 9 1 125 9 76.0 504 000

Low
7 10 5 4 375 28.75 74.4 501 000

Food supply in culture offilter-feeding organisms

275

high and control larval densities is given in Table 3. The total volume of filtered, heated, UV-sterilised sea water required to rear this number of larvae at high density is reduced by about 75% and labour involved in husbandry by about 70% compared with low-density rearing. The return in terms of spat produced is much the same. Fewer larval rearing vessels are required and consequently space is saved. Controlled feeding with the ACM in high-density rearing is not wasteful since only the volume of algae required to maintain the optimum food cell concentration for the surviving larvae is provided. In low-density rearing it is necessary to enrich a fixed volume of water to the optimum food cell density and this is independent of the number of surviving larvae. The calculation of the total volume of algae necessary to feed 1.24 million larvae to settlement at high density is detailed in Table 2. Cost in terms of food at a standard cell density was little different whether the larvae were grown at high or low densities (Table 3).

REFERENCES Haupt, K. (1979). A simple device to control concentrations of algae food in feeding experiments with filter feeding organisms. Ver6ff. Inst. Meeresforsch. Bremerhaven, 17,241-44. Helm, M. M. & Laing, I. (1981). Cost effective culture of marine unicellular algae. In: Energy Conservation and Use of Renewable Energies in the BioIndustries, ed. F. Vogt, Pergamon Press, Oxford and New York, pp. 247-59. Helm, M. M. & Millican, P. F. (1977). Experiments in the hatchery rearing of Pacific oyster larvae ( Crassostrea gigas Thunberg). Aquaculture, 11, 1-12. Munson, R. J. (1970). Turbidostats. In: Methods in Microbiology, Vol. 2, eds J. R. Norris and D. W. Ribbons, Academic Press, London and New York, pp. 347-76. Powell. E. O. (1963). Photometric methods in bacteriology. J. Sci. Food Agric.,
14, 1-8.

Walne. P. R. (1974). Culture of Bivalve Molluscs: 50 Years Experience at Conwy, Fishing News (Books), West Byfleet, 173 pp. Walne, P. R. & Helm, M. M. (1974). The routine culture of the Pacific oyster I Crassostrea gigas) at Conwy during 1973. Shellfish Inf. Leafl., Fish. Lab., Conw); No. 32.9 pp. Wickins, J. F. & Helm, M. M. (1981). Sea water treatment. In: Aquarium Systems, ed. A. D. Hawkins, Academic Press, London, pp. 63-128. Winter, J. E. (1973). The filtration rate of Mytilus edulis and its dependence on algal concentration measured by a continuous recording apparatus. Mar. Biol., 22, 317-28.