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An integrated system for microalgal and nursery seed clam culture
Aquacultural Engineering 24 (2000) 15 – 31 www.elsevier.nl/locate/aqua-online
An integrated system for microalgal and nursery seed clam culture Timothy J. Pfeiffer a,*, Kelly A. Rusch b a
Shellfish Aquaculture Laboratory, Uni6ersity of Georgia Marine Extension Ser6ice, 20 Ocean Science Circle, Sa6annah, GA 31411 -1011, USA b Department of Ci6il and En6ironmental Engineering, Louisiana State Uni6ersity, Baton Rouge, LA 70803 -6406, USA Received 10 October 1999; accepted 14 August 2000
Abstract A PC-controlled integrated system for the production of algae and culture of northern quahog seed was constructed. The diatom, Chaetoceros muelleri, was cultured in covered 550 l tanks. The harvested alga was the food source for the land-based nursery seed clam system. The nursery clam system consisted of six culture units constructed of clear PVC tubing, a 400 l feed reservoir, a solids separator, and a bead filter (0.03 m3). The culture units were 5 cm in diameter and 76 cm in height. The total system volume was 450 l and the recirculation flow rate was 40 lpm. Components of the computer control system include a laptop computer, a multiport connector, analog to digital converter (ADC) boards, and solid-state relays for each control output. Sampling, harvesting, refilling, and redosing of the algal chambers, seed clam feeding operations, cleaning of the seed bed by increased fluid flow, purging of the separator wastes, and backflushing of the bead filter were all computer controlled. The integrated system was tested using Mercenaria seed clams. The initial shell length of the seed clams were 2.5 (90.5) mm, and were stocked at a density of 3.0 g whole wet weight cm − 2. During 83 days of culture using the system the seed clams were sorted twice and reached an average shell length of 7.9 (9 0.8) mm. The percentage of survival percent was determined at different stages of growth during the culture period and ranged from 67.3 to 88.0%. © Published by Elsevier Science B.V. Keywords: Mercenaria mercenaria; Microalgae; Bivalve nursery; Recirculation
* Corresponding author. Present address: USDA-Agricultural Research Service, Aquaculture Systems Research Unit, University of Arkansas at Pine Bluff, 1200 N. University Dr, Department of Agriculture, Mail Slot 4912, Pine Bluff, AR 71601, USA. Tel.: +1-870-5438094; fax: +1-870-5438212. E-mail address: [email protected] (T.J. Pfeiffer). 0144-8609/00/$ - see front matter © Published by Elsevier Science B.V. PII: S 0 1 4 4 - 8 6 0 9 ( 0 0 ) 0 0 0 6 3 - 7
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1. Introduction The majority of the operational costs in a bivalve nursery are associated with algae culture and the maintenance (feeding and water quality) of seed stock (Castagna and Kraeuter, 1977; Coutteau et al., 1994). These costs can be reduced by increasing production per unit area or volume, minimizing labor requirements, and utilizing cultured microalgae more efficiently. The technologies of computercontrolled operation, water recirculation, and fluidization offer the potential to meet these objectives. These technologies offer the potential to reduce labor requirements, improve utilization efficiency of the algae produced, improve control and monitoring of system operation, reduce the solids and metabolic wastes in the seed culture system, sustain water quality for optimum seed growth, and reduced fouling of the culture units. A recent development of shellfish aquaculture is the high-density nursery culture of clutchless oysters (2 – 25 mm) by the use of water flowing upward at fluidization velocities (Ver and Wang, 1995). Under fluidized conditions, the bed of oysters is expanded due to the increased fluid flow. The oysters are suspended in the fluid rather than lying on each other as in a packed bed under low flow conditions. The fluidization allows for a more uniform distribution of the food supply and better transport of fecal material and other particulates out of the seed bed by the flowing water. Typical land-based clam nursery systems utilize upwellers for seed culture in which ambient seawater or seawater with cultured algae is pumped upward through the culture unit. The water flow through the upweller is too low for fluidization of seed to occur and as a result the density of seed is usually a single layer spread over the bottom of the upweller unit. In the last decade, the practice of culturing seed in a nursery system has flourished and the application of fluidized-bed technology coupled with computercontrol and recirculation technologies can potentially allow for the high-density culture of clam seed in a land-based nursery environment. An integrated system for the production of algae and culture of northern quahog seed clams, Mercenaria mercenaria, was developed utilizing computer-control, fluidization, and recirculation technologies. This paper presents (1) a description of the integrated system; (2) the computer-control strategy for the control and monitoring of the integrated system; and (3) system performance of culturing northern quahog seed clams.
2. Materials and methods The integrated system consists of three components (a) algal production unit; (b) seed clam culture unit; and (c) the computer control unit. A schematic outline of the integrated culture system is presented in Fig. 1 and a description of the individual components is provided below.
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2.1. Algal culture system The algae are grown in conical 550 l fiberglass tanks and covered with clear lexan to minimize the entrance of airborne contaminants. The microalgae, Chaetoceros muelleri (CHAET 10), was selected because it grows well at high temperatures and over a wide range of salinities (Johansen et al., 1990; Nelson et al., 1992). Feeding studies have also indicated CHAET 10 to be a suitable food source for Mercenaria seed (Wikfors et al., 1992; Walker et al., 1997). The air provided for culture aeration and mixing is supplied by a diaphragm air pump (Sweetwater, model L29). The air is filtered through an in-line filter capsule (0.2 microns) before entering the cultures. On a daily basis CO2 is continuously injected into the airline at a rate of 0.5 cm3 min − 1 for 6 h (1000 – 1600). Seawater for algal culture was obtained from the adjacent brackish river (Skidaway River). Treatment processes of the incoming seawater included bag filtration (20 and 5 micron), chlorination, active carbon filtration, UV sterilization, and ozonation. A one micron cartridge filter at the central inflow point of the culture tanks was the final water treatment step. Level sensors were used to control the water level in each tank. A float valve was placed into each tank to serve as the backup mechanism to the level sensors and prevent tank overflow. The millivolt output from a commercial turbidimeter (HACH 1720°C) was used to estimate the algal biomass (mg dry weight algae l − 1), in the culture tanks. During the computer-controlled sampling procedures miniature centrifugal pumps trans-
Fig. 1. Schematic diagram of the integrated algal production and recirculating seed clam nursery system at the Shellfish Aquaculture Laboratory, University of Georgia, Savannah, Georgia, USA.
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Fig. 2. General diagram of the upweller used in the recirculating seed clam nursery system for the fluidized-bed culture of Mercenaria seed clams.
ferred algal culture from the tanks to the turbidimeter at a rate of approximately 2 lpm. On the outside bottom of each tank was a 2.5 cm PVC tee, with 2.5 cm PVC ball valves fitted on both ends. One ball valve was connected to an actuator for controlling algal harvesting operations and the other ball valve was used to manually drain the tanks. A 0.076 kW (0.1 hp) centrifugal pump was computer-controlled to harvest the algal cultures and transfer the algae to a 500 l harvest reservoir. After the harvest procedure a peristaltic pump connected to the nutrient solutions (Fritz’s F/2 A and B) was activated to provided the dosing of nutrients into each tank.
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2.2. Seed clam nursery system The seed clam nursery system consisted of seven components (1) six 5-cm diameter clear PVC pipes, 76 cm in height, for seed clam culture (Fig. 2); (2) a 400-l system reservoir; (3) a 0.076 kW (0.1 hp) centrifugal pump for system water recirculation; (4) an in-line solids separator for removal of large particulate waste material; (5) a 0.028 m3 (1.0 ft3) bead filter for additional particle entrapment and nitrification; (6); a 0.37 kW (0.50 hp) chiller unit for water temperature control in the summer and (7); two header tubes. One tube served as the air escape vent from the bead filter after backflushing operations. The other tube provided static head to allow adjustments of water flow to each upweller to be made without affecting the adjusted flow in the other units. Algae for the clam system was obtained from the harvest reservoir of the algal culture system. Water loss in the clam system by evaporation or after filter backflushing was replaced by water from the water treatment system reservoir. Cleaning of the clam seed bed in the upweller units was done by the control program that closed the actuator valve at the end of the upweller line each hour for 30 s. Closing of this valve resulted in increase water flow through each of the upweller units thereby dislodging the seed mass clumps and flushing settled waste matter out of the seed bed. The control program also opened and closed valves for purging of wastes from the solids separator and for backflushing the bead filter. Each upweller contained a flow distribution plate (0.64 cm PVC with a radial pattern of 0.32 cm holes) to provide uniform water flow into the culture tubes. Placed slightly above the flow distribution plate was a mesh screen (1.0 mm Nytex) for retaining the seed mass. The water flow rate through the seed mass of each upweller was maintained and adjusted by an in-line flow meter placed at the inflow of each upweller. Flow through each upweller was manually adjusted with a PVC gate valve (1.9 cm) placed below the flow meter.
2.3. Process control The computer-control system included a laptop computer (Zenith Supersport e286), a multiport controller, three analog to digital converters (Remote Measurements Systems, Seattle, WA), and a 10-volt solid-state relay switch for each controlled output. The control program, was written in Turbo Pascal 6.0 (Borland International Inc., 1990). The program is menu driven to facilitate use by system users unfamiliar with the Turbo Pascal programming language. The program contained over 40 commands that are used to monitor or control all components of the integrated system. There were three program input parameters associated with the control and monitoring of the algal culture system. Two parameters were used to set the daily start (08:00 h) and end time (20:00 h) of algal culture sampling and harvesting operations. These times covered the range of observed algal growth under greenhouse conditions, where lighting for algal growth depends on the available sunlight. The third parameter was the biomass set point for algal harvest.
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Between the set hours of operation the chambers were sampled every 2 h to estimate the standing algal biomass. If the estimated algal biomass was lower than the harvest set point, the sampling procedure was complete and repeated in 2 h. If the estimated biomass was greater than the harvest set point, command actions were issued to the priority queue to initiate the harvest cycle. After harvesting a volume of 450 l, the chamber was refilled with the treated seawater and reinoculated with nutrients. The sampling procedure resumed 2 h after harvest. The recirculating nursery clam culture system had ten input parameters (1) initial clam biomass (g whole wet weight); (2) feeding rate (% of clam biomass per day); (3). estimated clam biomass growth rate (% per day); (4) feeding per day; (5). pump flow rate of algae from algal reservoir to clam reservoir (lpm); (6) algal reservoir concentration (mg l − 1 dry weight); (7) number of feedings between bead filter backflushing; (8) purge rate from solids separator (lpm); (9) frequency (min) in closing of the header valve; and (10) duration of header valve closure (s). The first eight parameters are used to calculate the daily dry weight of algae required for feeding the seed clams and to adjust the ration based on the estimated clam growth. The daily ration, adjusted at the end of each day (at 2345 h) according to the estimated growth rate, is calculated as a percent dry weight of algae per g whole wet weight of clams (% dry weight per wet weight per day) as follows: 1. Clam biomass (g), day n = [clam biomass (day per n)]× [estimated growth rate]. 2. Dry weight algae (g), day n =[clam biomass (day n)]× [% feed ration/100]. The feed ration delivered to the clam algae reservoir was then calculated as follows: 3. Ration amount, (g)=[Dry weight algae (g), day n]/number of feeding per day. The ration amount must be expressed volumetrically in order to determine the duration of the pump activation time to transfer algae from the algal harvest reservoir to the clam algal reservoir (the algal concentration in the calculation is the value of the harvest set point). 4. VA = [1/algal conc. (l mg − 1)] ×[ration amount (g algae)]× [1000 (mg g − 1)] 5. Pump time, Pt (min) = [VA (L)] ×[1/algal pump rate (lpm)]. Before this volume of algae can be pumped to the clam algal reservoir an equal volume must be removed from the clam system. Using the parameter value for the system purge rate (outflow from the swirl separator), the time interval required to displace an equal volume of clam system seawater via the separator purge valve was calculated: 6. Drain time (min) = [Pt (min)] × [algal pump rate per system purge rate]. The last calculation is the time interval between feeding. The input is in days and the program converts it to seconds to follow program control structure. This is calculated as follows: 7. Feed time interval (day)=[(24 h per day per feeding per day) × 3600 s h − 1]/ 86,400 s per day All of these calculations were performed as a single procedure before the feed command was initiated.
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2.4. Analytical procedures and techniques Control charts were maintained for the turbidimeter process of estimating the standing algal biomass and for the process of delivering algae from the algal harvest reservoir to the clam algal reservoir. A control chart is a graphical display of a specific characteristic that has been measured from a sample versus the sample number or time (Montgomery, 1997). The chart contains a centerline that represents the average value of the specific characteristic corresponding to the in-control state. The specific characteristic for the control chart of the recirculating seed clam nursery system was the amount of algae to be delivered (g dry weight). The characteristic for monitoring the turbidimeter was the harvest set point value. Two other horizontal lines on the chart, the upper control limit (UCL) and the lower control limit (LCL) are shown and are typically called the ‘3-sigma’ control limits. Sigma refers to the standard deviation (S.D.) of the statistic plotted on the chart not the S.D. of the quality characteristic. The processes were considered stable or in statistical control when the points plot within the control limits (X9 3s n) and no action is necessary. A point that plots outside the control limits is corrective action are required to find and eliminate the cause or causes responsible for this behavior. Samples for monitoring feeding operations and the turbidimeter were collected three times each week. Dry weight (mg l − 1) algal estimates were obtained from triplicate 100 ml samples. An ammonium formate solution (0.5 M) was used to prerinse the filters and the filtered algal sample to remove residual salts prior to oven drying at 100°C. The specific growth rate (SGR) of the seed clams in the culture units was calculated using the equation
SGR =ln
Nt /N0 ln(NA/N0) = dt dt
where N0, initial biomass of the seed clams in each upweller unit, g whole wet weight; Nt, final biomass of the seed clams in each upweller unit, g whole wet weight; dt, culture period, days. Percent seed bed expansion was obtained by the following equation: Percent seed bed expansion =
SVe 100, SVp
where, SVp, volume displacement of seed clam mass (ml); SVe, total volume displacement of expanded seed clam mass (ml).
2.5. Seed culture with the integrated system Hatchery reared (Mook Sea Farms, Damariscotta, Maine) northern quahog clam seed, M. mercenaria, of a single cohort were used in the 83-day trial period. The introductory trial was conducted during the fall and winter of 1996–1997. Initially, 40 ml (60 g whole wet weight) of seed (n= 12, 180) with a mean shell length of 2.5
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mm (S.D.9 0.5, n =300) was distributed into each upweller. During the trial period the seed clams in the culture upwellers units were thinned or sorted three times (day 14, 52, and 73). The initial stocking density was 3.0 g whole wet weight of seed cm-2 and was reduced to 2.0 – 3.0 g m − 2 after each sorting or thinning procedure. The feed ration and feeding frequency during the trial period is provided in Table 1. Based on the amount of available algae the daily ration ranged from 1 to 4% dry algae per wet weight clam during the trial period. A flow rate of 3.5 ( 90.3) lpm was maintained in each upweller during the trial period. A flow rate above 5.0 lpm moved the seed mass up the upweller column and out of the upweller unit. Water quality parameters of temperature, salinity, dissolved oxygen, and pH were measured in situ each morning. Levels of total ammonia-N, nitrite-N, nitrate-N, and total alkalinity were monitored weekly. pH measurements were obtained with a benchtop pH meter (Orion model 620), salinity with a hand-held, temperature compensated refractometer, and dissolved oxygen with a YSI oxygen meter (model Y58). Temperature probes in the clam system reservoir and algal chambers provided data acquired via the computer. Water samples for total ammonia-N, nitrite-N, nitrate-N, and alkalinity were analyzed using the HACH DREL2000 portable laboratory equipped with a DR 2000 direct reading spectrophotometer. Survival data was approximated for each individual upweller unit from the wet weight count of 1 g of clams.
3. Results
3.1. Algal culture During system operation the harvest set points for the algal cultures were between 30 and 60 mg l − 1 dry weight. In this range the sampling error of the HACH turbidimeter is approximately 25–13%, respectively. The control chart presented in Fig. 3 illustrates the performance of the HACH turbidimeter for measuring the algal tank culture biomass. A harvest set point of 40 mg l − 1 dry weight was used as the value of the center line in the control chart. The upper and lower limits of the control chart were 24.3 and 55.7 mg l − 1 dry weight, respectively, where sy =7.4 mg l − 1 and n = 2. Values were plotted in their sequence of measurement. Sample data points outside the control limits were a result of a biofilm on the sensor lens or algal floc in the sample rather than the turbidimeter being out of calibration. Consequently, part of the system daily maintenance was to clean the turbidimeter sensor lens and rinse the sample chamber. Fig. 4 presents a summer and winter algal production profile for Chaetoceros muelleri from the algal system under greenhouse conditions. The harvest levels were set at 60 mg l − 1 dry weight during spring and late summer seasons (March–October) and 30 mg l − 1 dry weight during the winter seasons (November–February). The harvest volume was 450 l, approximately 90% of the culture volume. A large harvest volume was employed since it was observed to minimize the growth of biofilm on the tank walls thereby extending the culture periods between tank
b
2 2 2 1 2 4 4
Daily feed rationa
0.5% 1.0% 1.0% 0.5% 1.0% 1.0% 1.0%
per per per per per per per
Feeding strategyb
6h 12 h 12 h 12 h 12 h 6h 6h
Percent of dry weight algae per wet weight clam. Percent of daily ration per feeding frequency.
6 6 6 6 6 4 2
1–14 14–42 42–52 52–59 59–73 73–83
a
Number of units
Days
3.0 2.0 2.7 2.5 2.7 3.0 3.0
Stocking density (g cm−2)
Ending density (g cm−2) 3.7 2.7 2.7 2.7 4.0 5.6 5.4
Initial shell length (mm)
2.5 9 0.5 2.7 9 0.6 3.1 90.8 3.6 9 0.5 3.8 9 0.5 4.2 9 0.5 5.7 9 0.5
2.7 90.6 3.1 90.8 3.1 90.8 3.8 90.5 4.4 90.8 5.59 0.7 7.9 90.8
Ending shell length (mm)
0.499 0.06 0.4990.05 0.4990.05 0.6790.02 0.6290.02 0.6490.01 0.679 0.01
Bed porosity
0.015 0.011 0.000 0.010 0.029 0.059 0.063
Mean daily growth rate (day−1)
Table 1 Trial results for Mercenaria seed clams grown in a land-based nursery system utilizing computer-control, recirculation, and fluidized-bed technology
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cleanings and reinoculation. In addition, the large harvest volumes helped control the protozoan and bacterial contamination of the algal culture as the harvested culture volume was replaced with the relatively contaminant-free filtered seawater. Sunlight availability and photoperiod, as well as the ambient temperature in the greenhouse principally governed the harvest set point during system operation. In the summer months, the high ambient air temperature inside the greenhouse resulted in the water temperature in the closed culture chambers to reach levels (\ 35°C) considered sub-optimum for growth (Chen, 1991). To minimize algal cultures collapsing from the high temperatures, the chambers were batch-harvested approximately every 3 days following the initial harvest. In the summer, refilling the chambers after harvesting with the chilled reservoir seawater (20°C) of the water treatment unit was a helpful passive approach for maintaining the culture temperatures below 35°C during the 3-day culture period. With this harvest frequency (3 days), a 90% harvest of the culture, and avoidance of high temperatures, it was difficult to maintain a harvest biomass level above 60 mg l − 1 dry weight. In the winter, the reduced sunlight and the low ambient temperatures in the greenhouse limited biomass growth. By lengthening the harvest intervals to 4 days a culture biomass level of 30 mg l − 1 dry weight was attainable. Beyond a 4-day culture period minimal additional growth was observed.
Fig. 3. Example of the control chart maintained for the HACH 1720C turbidimeter measuring process of estimating the culture chamber algal biomass.
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Fig. 4. Profile of the growth for CHAET 10 in the algal chambers under greenhouse conditions during the summer and winter months.
3.2. Control operations of algal deli6ery and nursery system There were several concerns with regards to delivering algae from the algae production unit to the seed clam nursery unit. The major concern was the error associated with estimating the alga culture biomass. Underestimating the algal biomass can result in the clams being overfed and inefficient use of the algae produced. An overestimate can result in the underfeeding of the seed clams thus lowering growth rates and potentially increasing the mortality rates. Thus, a cost-effective approach was required for estimating the algal biomass. The low range turbidimeter provided acceptable performance and stability, as indicated by the control chart (Fig. 3), for estimating the algal biomass. Utilizing an in-line fluorimeter (approximately 10× more expensive than the turbidimeter) was not considered cost-effective for the minimal gain in performance (approximately 5%). Utilization of an algal biomass sensor comprised of a photovoltaic solarcell and light source, and much less expensive than the turbidimeter (100× less expensive) was rejected as the unit required constant calibration and performance reliability was inconsistent. Another concern was with the computer-controlled volume exchanges between the algal and seed clam system. The volume of water discharged from the seed clam unit by backflushing the beadfilter or purging the solids separator was replaced by
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delivering an equal volume of algae from the algal system. The computer-controlled volume exchanges were within the control chart UCL and LCL limits and changes to the delivery setup were not necessary (i.e. adjustment of the algal pump activation time period or reservoir purge time period). There was also a concern with the cell integrity and viability from the shear stress in using centrifugal pumps for harvesting, transferring and recirculating the microalgae. Microscope observation indicated cell walls remained intact and no difference in whole cell counts was observed before and after harvesting algae from the culture chambers or after transfer from the algal harvest reservoir to the clam system reservoir. Additionally, when recirculating the algae in the nursery system without seed clams present and bypassing the beadfilter, cell counts and suspended solids analysis did not indicate a reduction in cell numbers or biomass. Another reason for the concern with regards to accurate estimates of algal biomass and volume exchanges was the need to avoid a high algal concentration in the seed clam system. A high algal concentration in a seed clam system can result in pseudofeces production and inefficient algal utilization (Tenore and Dunstan, 1973). Pseudofeces production represents a loss of potentially utilizable algal cells and the production of organic matter in the form of mucous. Consequently, fouling of the seed bed increases which minimizes uniform water flow and food distribution through the seed bed. All of these conditions result in the decrease of seed clam growth. Therefore, a pulsed feeding strategy was utilized to avoid high cell concentrations in the system and minimize psuedofeces production. The transfer of algae from the algal system harvest reservoir to the clam feed reservoir was 2–4 times a day, depending on the alga concentration and availability. The resulting algal concentration in the seed clam system after each feed transfer ranged from 50 000 to 200 000 cells ml − 1. As stated above, before each feeding, a volume of water equal to the volume of algae being transferred was removed from the seed clam system. This volume of water, removed by backflushing the bead filter or by purging the solids separator, resulted in a water volume exchange per feeding from 11 to 40%. The resulting total daily volume exchange ranged from 20 to 160%. With such high daily system volume exchange, incorporating the biofilter into the recirculation process is not necessary and eliminating the biofilter would reduce any algal ration loss due to filter entrapment.
3.3. Culture of seed clams The growth of the seed clams in the integrated system cultured under high-density, fluidized-flow conditions is presented in Fig. 5. The culture trial was terminated after 83 days, due to a sustained decline in the ambient air temperature of the greenhouse resulting in sub-optimal culture unit water temperature for seed clam growth and insufficient cultured algae for a food supply. The mean daily and weekly water quality data are summarized in Table 2. There were no major differences in the progression of the water quality parameters with time during the culture period except for water temperature. The water temperature for the seed
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Fig. 5. Profile of the growth for Mercenaria seed clams utilizing fluidized flow in a computer-controlled integrated algal-seed clam nursery system.
clam system progressively declined due to the seasonal changes in temperature and lack of temperature control in the greenhouse. After 2 weeks of culture from initial stocking, seed survival was 76.8% ( 9 11.5%) with an unexpectedly low growth rate (0.015 per day). Compared to previous laboratory data for forced-flow upweller culture of Mercenaria seed, the growth rate was approximately 70% lower. The stocking density was thus thinned from 3.7 g wet weight clam cm − 2 to 2.0 g wet weight clam cm − 2. After an additional four Table 2 Water quality results of the land-based nursery system utilizing recirculation and fluidized-flow technology Variable
Average
Temperature (°C) 19.0 Dissolved oxygen (mg l−1) 7.2 pH 7.5 Salinity (ppt) 26.0 Ammonia-N (mg l−1) 0.005 Nitrite-N (mg l−1) 0.27 Nitrate-N (mg l−1) 14.1 Total alkalinity (mg l−1 CaCO3) 103.1
Standard deviation
Maximum value Minimum value
1.8 0.6 0.2 1.1 0.017 0.55 5.8 10.3
28.0 9.0 7.9 28.0 0.090 2.30 26.4 121.0
16.6 6.0 7.3 24.0 0.000 0.01 3.8 70.0
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and a half weeks of culture, minimal growth (0.4 mm shell length) of the seed clams was observed and survival rates were lower, 67.3% (9 6.7%). During this 52-day culture period, there was noticeable byssal thread attachment among the smaller clams in the upweller culture units. To minimize and breakup the byssal thread attachment amongst the smaller seed, the actuator valve to the second header tube was closed for 1 min every hour. The resulting increased water flow through each upweller (approximately 25% increase) helped breakup seed clumping, but was not sufficient. It is interesting to note that some of the air which was displaced after the beadfilter was backflushed escaped into the upwellers units instead of through the header tube and was more effective at expanding and breaking up the seed mass, and clearing settled solids from the seed bed. As a result of the byssal thread attachment amongst the smaller seed in the culture units, seed less than 3.0 mm in shell length were removed. The mean shell length of the seed replaced in the upwellers was 3.6 mm (S.D. 9 0.5) and the resulting bed porosity increased from 0.49 to 0.67. A week after the small seed were removed (day 59), the growth rate of the seed improved from 0 to 0.01 per day. On day 73, 3 weeks after the removal of the small seed, the mean shell length increased from 3.6 ( 90.5) to 4.4 ( 90.8) mm. The growth rate tripled to 0.029 per day and survival rate improved to 88.0% (9 1.7%). The shell length measurements indicated a wide distribution of seed size, therefore, a second sorting was conducted to separate the larger seed from the smaller seed. The larger seed, those retained on a 5 mm sieve, were distributed into two upwellers at a density of 3.0 g wet weight cm − 2. The remaining seed, those retained on a 3 mm sieve, were distributed into the remaining four upwellers at the same density. The trial period was terminated 10 days later because sufficient quantities of cultured algae for the seed clams were unavailable and declining ambient temperature inside the greenhouse. During the last 10 days of culture, the average growth rates between the two groups improved to 0.059– 0.063 per day, respectively. Survival rates dropped to 78.1% (9 6.5%) for the units with the smaller seed and 72.0% (9 4.4%) for those with the larger seed. The handling stress from the sorting procedures and lower water temperatures may have potentially resulted in the lower survival rates. Once the smaller seed were removed on day 52 from the culture units, the bed porosity remained steady, ranging from 0.62 to 0.67. The trial results indicating growth rates, bed porosity, shell length, stocking densities, feeding rations and strategies are summarized in Table 1. 4. Discussion The integrated control system performed dependably, sampling and harvesting the alga culture chambers as required, estimating the alga biomass and delivering the calculated volume of algal food within the control limits, and purging the solids separator and backflushing the bead filter as directed. During the trial period there were no valve or pump malfunctions or computer failures resulting in inactivity of control procedures. However, more operating time is needed to evaluate the durability of the system.
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Automating the production of algae provides an interesting alternative to the current manual batch culture method for algae culture at the laboratory and offers the potential to reduce the daily culture labor activities. The majority of the labor savings (approximately 30%) were experienced in reducing the time involved with maintaining and harvesting cultures of different volumes (2 and 10 l carboys, 200 l kalwalls). Furthermore, the algae produced by the automated system were quantified, thus permitting efficient use of the algae when being feed to the seed clams. In addition to employing computer-control to limit labor needs another objective was to provide the Mercenaria seed clams with a dependable and consistent source of algal food for maintaining growth. A ‘crash’ or contamination of the algal culture can result in a loss of a large investment in the animals. Consequently, part of the harvest strategy was to withdraw the maximum amount of usable algae from the system when the desired culture phase was attained. There were constraints that limited consistent and reliable long-term algal production. The primary constraint was operating under ambient greenhouse conditions (variable light and temperature). Other significant culture constraints were bacteria and protozoan contamination, and the biofilm buildup on the tank walls, particularly in the summer. A large harvest volume combined with refilling harvested cultures with treated seawater helped reduced culture contamination and biofilm growth. This batch culture procedure extended culture operation to approximately three weeks before tank cleanings and reinoculation with fresh alga cultures were necessary. The control program was modified to allow the removal of an algal culture tank from system operation for this management process. This was perceived as a more practical method than trying to maintain sterility of large algal cultures on a continual basis, which may not be achievable on a commercial clam operation because of the expense. Fluidization of the seed bed may be a potential culture technique for the high-density land-based nursery culture of Mercenaria seed. Typical seed clam culture at the laboratory involves placing a single layer of seed (initial stocking density approximately 0.2 g wet weight cm − 2) into the upweller culture units and using a low water flow rate to pass cultured algae through the seed mass. By increasing the flow to fluidization velocities in the upwellers, the initial seed stocking density was increased more than 10-fold (3.0 g wet weight cm − 2). However, for smaller seed (B3-mm shell length), the fluidized-flow conditions induced greater byssal thread attachment amongst the seed. The bysall thread attachment caused clumping of the seed mass, thereby affecting water flow and food distribution and reducing uniform seed growth. The clumping of the seed mass was minimized and the seed bed cleaned of settled solids by passing air through the seed mass. Incorporating an air scour of the seed mass versus the hydraulic expansion warrants further evaluation especially if culture conditions involve high densities and fluidization of small size seed. Additionally, a pulse feeding strategy was incorporated to limit the food concentration to the seed, because a high feeding level can result in the production of psuedofeces and inefficient utilization of the feed. When a 4% daily ration was
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provided and divided into four feedings per day, psuedofece production was not observed and over 90% of the algal feed was cleared from suspension within the 6 h feeding time. However, the need for a daily ration greater than the recommended 1.5–2% (Coutteau et al., 1994) appeared to be a result of particle entrapment by the bead filter since as much as 50% of the supplied algal feed was observed to be removed by the bead filter. The daily exchange of the system water volume was high, 40– 160% of the seed clam system volume. This high water exchange volume offsets the need of the bead filter for ammonia removal and reduces alga losses by filter entrapment, which could otherwise be available for the seed. The approximate total cost of the integrated system was $23,100. This cost included the purchase of the personal computer, software, and all components for the water treatment, alga culture, clam culture, and computer-control units. All components of the system described here are readily available and the expense for each system unit was 28.4, 21.5, 25.3, and 24.8%, respectively. With greater availability of alga products (pastes and dried matter) and computer control materials, there are a number of options which commercial facilities may utilized for system control at considerable lower costs. Low-cost programmable logic controllers (about US $100 and up) or single board computers (about US $300 and up) programmed to delivery a premeasured alga paste solution may offer commercial facilities the benefits of optimized, automated feeding system for shellfish without the need of an alga production or water treatment units. The automated feeding of alga paste material warrants further investigation for significant technological advancement to culture systems.
Acknowledgements This work was funded by the University of Georgia Marine Extension Service and the Georgia Sea Grant College Program under grant number NA66RG0282. The Georgia Sea Grant College Program is an element of the National Sea Grant College Program under the direction of National Oceanic and Atmospheric Administration, U.S. Department of Commerce. The authors thank Dr Randal Walker, Dr James Nelson, and Charles Robertson, and other staff at the University of Georgia Shellfish Aquaculture Laboratory and Skidaway Institute of Oceanography for use of facilities and assistance during the system operation and evaluation. The authors also thank Dr Steve Jodis of Armstrong Atlantic State University for his assistance with control program modifications. Mention of a specific product or trade name does not constitute an endorsement nor imply its approval to the exclusion of other suitable products.
References Borland International Inc., 1990. Turbo Pascal User’s Guide, Version 6.0. Scotts Valley, CA 95067-0001
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Castagna, M.A., Kraeuter, J.N., 1977. Mercenaria culture using stone aggregate for predator protection. Proc. Nat. Shellfish Assoc. 67, 1–6. Chen, J.F. 1991. Commercial production of microalgae and rotifers in China. In: W. and K.L. Main (Eds.) Rotifer and Microalgae Culture Systems. Proceedings of a U.S.-Asia Workshop. Fulks. Oceanic Institute, Honolulu, HI, pp. 105–112. Coutteau, P., Hadley, N.H., Manzi, J.J., Sorgeloos, P., 1994. Effect of algal ration and substitution of algae by manipulated yeast diets on the growth of juvenile Mercenaria mercenaria. Aquaculture 120, 135 – 150. Johansen, J.R., Braclay, W.R., Nagle, N., 1990. Physiological variability within ten strains of Chaetoceros muelleri (Bacillariophyceae). J. Phycol. 26, 271 – 278. Montgomery, D.C., 1997. Design and Analysis of Experiments, fourth ed. Wiley, New York. Nelson, J.R., Guardia, S., Cowell, L.E., Heffernan, P.B., 1992. Evaluation of microalgae clones for mass culture in a subtropical greenhouse hatchery: growth rates and biochemical composition at 30°C. Aquaculture 106, 357–377. Tenore, K.R., Dunstan, W.M., 1973. Comparison of feeding and biodeposition of three bivalves at different food levels. Marine Biol. 21, 190–195. Ver, L.M.B., Wang, J.K., 1995. Design criteria of a fluidized bed oyster nursery. Aquac. Eng. 14, 229 – 249. Walker, R.W., Heffernan, P.B., Hurley, D.H., 1997. Growth of Mercenaria mercenaria larvae and juveniles fed two microalgae species under greenhouse conditions in coastal Georgia. Georgia J. Sci. 55, 407 –411. Wikfors, G.H., Ferris, G.E., Smith, B.C., 1992. The relationship between gross biochemical composition of cultured algal foods and growth of the hard clam, Mercenaria mercenaria (L.). Aquaculture 108, 135 – 154.
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An integrated system for microalgal and nursery seed clam culture Timothy J. Pfeiffer a,*, Kelly A. Rusch b a
Shellfish Aquaculture Laboratory, Uni6ersity of Georgia Marine Extension Ser6ice, 20 Ocean Science Circle, Sa6annah, GA 31411 -1011, USA b Department of Ci6il and En6ironmental Engineering, Louisiana State Uni6ersity, Baton Rouge, LA 70803 -6406, USA Received 10 October 1999; accepted 14 August 2000
Abstract A PC-controlled integrated system for the production of algae and culture of northern quahog seed was constructed. The diatom, Chaetoceros muelleri, was cultured in covered 550 l tanks. The harvested alga was the food source for the land-based nursery seed clam system. The nursery clam system consisted of six culture units constructed of clear PVC tubing, a 400 l feed reservoir, a solids separator, and a bead filter (0.03 m3). The culture units were 5 cm in diameter and 76 cm in height. The total system volume was 450 l and the recirculation flow rate was 40 lpm. Components of the computer control system include a laptop computer, a multiport connector, analog to digital converter (ADC) boards, and solid-state relays for each control output. Sampling, harvesting, refilling, and redosing of the algal chambers, seed clam feeding operations, cleaning of the seed bed by increased fluid flow, purging of the separator wastes, and backflushing of the bead filter were all computer controlled. The integrated system was tested using Mercenaria seed clams. The initial shell length of the seed clams were 2.5 (90.5) mm, and were stocked at a density of 3.0 g whole wet weight cm − 2. During 83 days of culture using the system the seed clams were sorted twice and reached an average shell length of 7.9 (9 0.8) mm. The percentage of survival percent was determined at different stages of growth during the culture period and ranged from 67.3 to 88.0%. © Published by Elsevier Science B.V. Keywords: Mercenaria mercenaria; Microalgae; Bivalve nursery; Recirculation
* Corresponding author. Present address: USDA-Agricultural Research Service, Aquaculture Systems Research Unit, University of Arkansas at Pine Bluff, 1200 N. University Dr, Department of Agriculture, Mail Slot 4912, Pine Bluff, AR 71601, USA. Tel.: +1-870-5438094; fax: +1-870-5438212. E-mail address: [email protected] (T.J. Pfeiffer). 0144-8609/00/$ - see front matter © Published by Elsevier Science B.V. PII: S 0 1 4 4 - 8 6 0 9 ( 0 0 ) 0 0 0 6 3 - 7
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1. Introduction The majority of the operational costs in a bivalve nursery are associated with algae culture and the maintenance (feeding and water quality) of seed stock (Castagna and Kraeuter, 1977; Coutteau et al., 1994). These costs can be reduced by increasing production per unit area or volume, minimizing labor requirements, and utilizing cultured microalgae more efficiently. The technologies of computercontrolled operation, water recirculation, and fluidization offer the potential to meet these objectives. These technologies offer the potential to reduce labor requirements, improve utilization efficiency of the algae produced, improve control and monitoring of system operation, reduce the solids and metabolic wastes in the seed culture system, sustain water quality for optimum seed growth, and reduced fouling of the culture units. A recent development of shellfish aquaculture is the high-density nursery culture of clutchless oysters (2 – 25 mm) by the use of water flowing upward at fluidization velocities (Ver and Wang, 1995). Under fluidized conditions, the bed of oysters is expanded due to the increased fluid flow. The oysters are suspended in the fluid rather than lying on each other as in a packed bed under low flow conditions. The fluidization allows for a more uniform distribution of the food supply and better transport of fecal material and other particulates out of the seed bed by the flowing water. Typical land-based clam nursery systems utilize upwellers for seed culture in which ambient seawater or seawater with cultured algae is pumped upward through the culture unit. The water flow through the upweller is too low for fluidization of seed to occur and as a result the density of seed is usually a single layer spread over the bottom of the upweller unit. In the last decade, the practice of culturing seed in a nursery system has flourished and the application of fluidized-bed technology coupled with computercontrol and recirculation technologies can potentially allow for the high-density culture of clam seed in a land-based nursery environment. An integrated system for the production of algae and culture of northern quahog seed clams, Mercenaria mercenaria, was developed utilizing computer-control, fluidization, and recirculation technologies. This paper presents (1) a description of the integrated system; (2) the computer-control strategy for the control and monitoring of the integrated system; and (3) system performance of culturing northern quahog seed clams.
2. Materials and methods The integrated system consists of three components (a) algal production unit; (b) seed clam culture unit; and (c) the computer control unit. A schematic outline of the integrated culture system is presented in Fig. 1 and a description of the individual components is provided below.
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2.1. Algal culture system The algae are grown in conical 550 l fiberglass tanks and covered with clear lexan to minimize the entrance of airborne contaminants. The microalgae, Chaetoceros muelleri (CHAET 10), was selected because it grows well at high temperatures and over a wide range of salinities (Johansen et al., 1990; Nelson et al., 1992). Feeding studies have also indicated CHAET 10 to be a suitable food source for Mercenaria seed (Wikfors et al., 1992; Walker et al., 1997). The air provided for culture aeration and mixing is supplied by a diaphragm air pump (Sweetwater, model L29). The air is filtered through an in-line filter capsule (0.2 microns) before entering the cultures. On a daily basis CO2 is continuously injected into the airline at a rate of 0.5 cm3 min − 1 for 6 h (1000 – 1600). Seawater for algal culture was obtained from the adjacent brackish river (Skidaway River). Treatment processes of the incoming seawater included bag filtration (20 and 5 micron), chlorination, active carbon filtration, UV sterilization, and ozonation. A one micron cartridge filter at the central inflow point of the culture tanks was the final water treatment step. Level sensors were used to control the water level in each tank. A float valve was placed into each tank to serve as the backup mechanism to the level sensors and prevent tank overflow. The millivolt output from a commercial turbidimeter (HACH 1720°C) was used to estimate the algal biomass (mg dry weight algae l − 1), in the culture tanks. During the computer-controlled sampling procedures miniature centrifugal pumps trans-
Fig. 1. Schematic diagram of the integrated algal production and recirculating seed clam nursery system at the Shellfish Aquaculture Laboratory, University of Georgia, Savannah, Georgia, USA.
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Fig. 2. General diagram of the upweller used in the recirculating seed clam nursery system for the fluidized-bed culture of Mercenaria seed clams.
ferred algal culture from the tanks to the turbidimeter at a rate of approximately 2 lpm. On the outside bottom of each tank was a 2.5 cm PVC tee, with 2.5 cm PVC ball valves fitted on both ends. One ball valve was connected to an actuator for controlling algal harvesting operations and the other ball valve was used to manually drain the tanks. A 0.076 kW (0.1 hp) centrifugal pump was computer-controlled to harvest the algal cultures and transfer the algae to a 500 l harvest reservoir. After the harvest procedure a peristaltic pump connected to the nutrient solutions (Fritz’s F/2 A and B) was activated to provided the dosing of nutrients into each tank.
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2.2. Seed clam nursery system The seed clam nursery system consisted of seven components (1) six 5-cm diameter clear PVC pipes, 76 cm in height, for seed clam culture (Fig. 2); (2) a 400-l system reservoir; (3) a 0.076 kW (0.1 hp) centrifugal pump for system water recirculation; (4) an in-line solids separator for removal of large particulate waste material; (5) a 0.028 m3 (1.0 ft3) bead filter for additional particle entrapment and nitrification; (6); a 0.37 kW (0.50 hp) chiller unit for water temperature control in the summer and (7); two header tubes. One tube served as the air escape vent from the bead filter after backflushing operations. The other tube provided static head to allow adjustments of water flow to each upweller to be made without affecting the adjusted flow in the other units. Algae for the clam system was obtained from the harvest reservoir of the algal culture system. Water loss in the clam system by evaporation or after filter backflushing was replaced by water from the water treatment system reservoir. Cleaning of the clam seed bed in the upweller units was done by the control program that closed the actuator valve at the end of the upweller line each hour for 30 s. Closing of this valve resulted in increase water flow through each of the upweller units thereby dislodging the seed mass clumps and flushing settled waste matter out of the seed bed. The control program also opened and closed valves for purging of wastes from the solids separator and for backflushing the bead filter. Each upweller contained a flow distribution plate (0.64 cm PVC with a radial pattern of 0.32 cm holes) to provide uniform water flow into the culture tubes. Placed slightly above the flow distribution plate was a mesh screen (1.0 mm Nytex) for retaining the seed mass. The water flow rate through the seed mass of each upweller was maintained and adjusted by an in-line flow meter placed at the inflow of each upweller. Flow through each upweller was manually adjusted with a PVC gate valve (1.9 cm) placed below the flow meter.
2.3. Process control The computer-control system included a laptop computer (Zenith Supersport e286), a multiport controller, three analog to digital converters (Remote Measurements Systems, Seattle, WA), and a 10-volt solid-state relay switch for each controlled output. The control program, was written in Turbo Pascal 6.0 (Borland International Inc., 1990). The program is menu driven to facilitate use by system users unfamiliar with the Turbo Pascal programming language. The program contained over 40 commands that are used to monitor or control all components of the integrated system. There were three program input parameters associated with the control and monitoring of the algal culture system. Two parameters were used to set the daily start (08:00 h) and end time (20:00 h) of algal culture sampling and harvesting operations. These times covered the range of observed algal growth under greenhouse conditions, where lighting for algal growth depends on the available sunlight. The third parameter was the biomass set point for algal harvest.
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Between the set hours of operation the chambers were sampled every 2 h to estimate the standing algal biomass. If the estimated algal biomass was lower than the harvest set point, the sampling procedure was complete and repeated in 2 h. If the estimated biomass was greater than the harvest set point, command actions were issued to the priority queue to initiate the harvest cycle. After harvesting a volume of 450 l, the chamber was refilled with the treated seawater and reinoculated with nutrients. The sampling procedure resumed 2 h after harvest. The recirculating nursery clam culture system had ten input parameters (1) initial clam biomass (g whole wet weight); (2) feeding rate (% of clam biomass per day); (3). estimated clam biomass growth rate (% per day); (4) feeding per day; (5). pump flow rate of algae from algal reservoir to clam reservoir (lpm); (6) algal reservoir concentration (mg l − 1 dry weight); (7) number of feedings between bead filter backflushing; (8) purge rate from solids separator (lpm); (9) frequency (min) in closing of the header valve; and (10) duration of header valve closure (s). The first eight parameters are used to calculate the daily dry weight of algae required for feeding the seed clams and to adjust the ration based on the estimated clam growth. The daily ration, adjusted at the end of each day (at 2345 h) according to the estimated growth rate, is calculated as a percent dry weight of algae per g whole wet weight of clams (% dry weight per wet weight per day) as follows: 1. Clam biomass (g), day n = [clam biomass (day per n)]× [estimated growth rate]. 2. Dry weight algae (g), day n =[clam biomass (day n)]× [% feed ration/100]. The feed ration delivered to the clam algae reservoir was then calculated as follows: 3. Ration amount, (g)=[Dry weight algae (g), day n]/number of feeding per day. The ration amount must be expressed volumetrically in order to determine the duration of the pump activation time to transfer algae from the algal harvest reservoir to the clam algal reservoir (the algal concentration in the calculation is the value of the harvest set point). 4. VA = [1/algal conc. (l mg − 1)] ×[ration amount (g algae)]× [1000 (mg g − 1)] 5. Pump time, Pt (min) = [VA (L)] ×[1/algal pump rate (lpm)]. Before this volume of algae can be pumped to the clam algal reservoir an equal volume must be removed from the clam system. Using the parameter value for the system purge rate (outflow from the swirl separator), the time interval required to displace an equal volume of clam system seawater via the separator purge valve was calculated: 6. Drain time (min) = [Pt (min)] × [algal pump rate per system purge rate]. The last calculation is the time interval between feeding. The input is in days and the program converts it to seconds to follow program control structure. This is calculated as follows: 7. Feed time interval (day)=[(24 h per day per feeding per day) × 3600 s h − 1]/ 86,400 s per day All of these calculations were performed as a single procedure before the feed command was initiated.
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2.4. Analytical procedures and techniques Control charts were maintained for the turbidimeter process of estimating the standing algal biomass and for the process of delivering algae from the algal harvest reservoir to the clam algal reservoir. A control chart is a graphical display of a specific characteristic that has been measured from a sample versus the sample number or time (Montgomery, 1997). The chart contains a centerline that represents the average value of the specific characteristic corresponding to the in-control state. The specific characteristic for the control chart of the recirculating seed clam nursery system was the amount of algae to be delivered (g dry weight). The characteristic for monitoring the turbidimeter was the harvest set point value. Two other horizontal lines on the chart, the upper control limit (UCL) and the lower control limit (LCL) are shown and are typically called the ‘3-sigma’ control limits. Sigma refers to the standard deviation (S.D.) of the statistic plotted on the chart not the S.D. of the quality characteristic. The processes were considered stable or in statistical control when the points plot within the control limits (X9 3s n) and no action is necessary. A point that plots outside the control limits is corrective action are required to find and eliminate the cause or causes responsible for this behavior. Samples for monitoring feeding operations and the turbidimeter were collected three times each week. Dry weight (mg l − 1) algal estimates were obtained from triplicate 100 ml samples. An ammonium formate solution (0.5 M) was used to prerinse the filters and the filtered algal sample to remove residual salts prior to oven drying at 100°C. The specific growth rate (SGR) of the seed clams in the culture units was calculated using the equation
SGR =ln
Nt /N0 ln(NA/N0) = dt dt
where N0, initial biomass of the seed clams in each upweller unit, g whole wet weight; Nt, final biomass of the seed clams in each upweller unit, g whole wet weight; dt, culture period, days. Percent seed bed expansion was obtained by the following equation: Percent seed bed expansion =
SVe 100, SVp
where, SVp, volume displacement of seed clam mass (ml); SVe, total volume displacement of expanded seed clam mass (ml).
2.5. Seed culture with the integrated system Hatchery reared (Mook Sea Farms, Damariscotta, Maine) northern quahog clam seed, M. mercenaria, of a single cohort were used in the 83-day trial period. The introductory trial was conducted during the fall and winter of 1996–1997. Initially, 40 ml (60 g whole wet weight) of seed (n= 12, 180) with a mean shell length of 2.5
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mm (S.D.9 0.5, n =300) was distributed into each upweller. During the trial period the seed clams in the culture upwellers units were thinned or sorted three times (day 14, 52, and 73). The initial stocking density was 3.0 g whole wet weight of seed cm-2 and was reduced to 2.0 – 3.0 g m − 2 after each sorting or thinning procedure. The feed ration and feeding frequency during the trial period is provided in Table 1. Based on the amount of available algae the daily ration ranged from 1 to 4% dry algae per wet weight clam during the trial period. A flow rate of 3.5 ( 90.3) lpm was maintained in each upweller during the trial period. A flow rate above 5.0 lpm moved the seed mass up the upweller column and out of the upweller unit. Water quality parameters of temperature, salinity, dissolved oxygen, and pH were measured in situ each morning. Levels of total ammonia-N, nitrite-N, nitrate-N, and total alkalinity were monitored weekly. pH measurements were obtained with a benchtop pH meter (Orion model 620), salinity with a hand-held, temperature compensated refractometer, and dissolved oxygen with a YSI oxygen meter (model Y58). Temperature probes in the clam system reservoir and algal chambers provided data acquired via the computer. Water samples for total ammonia-N, nitrite-N, nitrate-N, and alkalinity were analyzed using the HACH DREL2000 portable laboratory equipped with a DR 2000 direct reading spectrophotometer. Survival data was approximated for each individual upweller unit from the wet weight count of 1 g of clams.
3. Results
3.1. Algal culture During system operation the harvest set points for the algal cultures were between 30 and 60 mg l − 1 dry weight. In this range the sampling error of the HACH turbidimeter is approximately 25–13%, respectively. The control chart presented in Fig. 3 illustrates the performance of the HACH turbidimeter for measuring the algal tank culture biomass. A harvest set point of 40 mg l − 1 dry weight was used as the value of the center line in the control chart. The upper and lower limits of the control chart were 24.3 and 55.7 mg l − 1 dry weight, respectively, where sy =7.4 mg l − 1 and n = 2. Values were plotted in their sequence of measurement. Sample data points outside the control limits were a result of a biofilm on the sensor lens or algal floc in the sample rather than the turbidimeter being out of calibration. Consequently, part of the system daily maintenance was to clean the turbidimeter sensor lens and rinse the sample chamber. Fig. 4 presents a summer and winter algal production profile for Chaetoceros muelleri from the algal system under greenhouse conditions. The harvest levels were set at 60 mg l − 1 dry weight during spring and late summer seasons (March–October) and 30 mg l − 1 dry weight during the winter seasons (November–February). The harvest volume was 450 l, approximately 90% of the culture volume. A large harvest volume was employed since it was observed to minimize the growth of biofilm on the tank walls thereby extending the culture periods between tank
b
2 2 2 1 2 4 4
Daily feed rationa
0.5% 1.0% 1.0% 0.5% 1.0% 1.0% 1.0%
per per per per per per per
Feeding strategyb
6h 12 h 12 h 12 h 12 h 6h 6h
Percent of dry weight algae per wet weight clam. Percent of daily ration per feeding frequency.
6 6 6 6 6 4 2
1–14 14–42 42–52 52–59 59–73 73–83
a
Number of units
Days
3.0 2.0 2.7 2.5 2.7 3.0 3.0
Stocking density (g cm−2)
Ending density (g cm−2) 3.7 2.7 2.7 2.7 4.0 5.6 5.4
Initial shell length (mm)
2.5 9 0.5 2.7 9 0.6 3.1 90.8 3.6 9 0.5 3.8 9 0.5 4.2 9 0.5 5.7 9 0.5
2.7 90.6 3.1 90.8 3.1 90.8 3.8 90.5 4.4 90.8 5.59 0.7 7.9 90.8
Ending shell length (mm)
0.499 0.06 0.4990.05 0.4990.05 0.6790.02 0.6290.02 0.6490.01 0.679 0.01
Bed porosity
0.015 0.011 0.000 0.010 0.029 0.059 0.063
Mean daily growth rate (day−1)
Table 1 Trial results for Mercenaria seed clams grown in a land-based nursery system utilizing computer-control, recirculation, and fluidized-bed technology
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cleanings and reinoculation. In addition, the large harvest volumes helped control the protozoan and bacterial contamination of the algal culture as the harvested culture volume was replaced with the relatively contaminant-free filtered seawater. Sunlight availability and photoperiod, as well as the ambient temperature in the greenhouse principally governed the harvest set point during system operation. In the summer months, the high ambient air temperature inside the greenhouse resulted in the water temperature in the closed culture chambers to reach levels (\ 35°C) considered sub-optimum for growth (Chen, 1991). To minimize algal cultures collapsing from the high temperatures, the chambers were batch-harvested approximately every 3 days following the initial harvest. In the summer, refilling the chambers after harvesting with the chilled reservoir seawater (20°C) of the water treatment unit was a helpful passive approach for maintaining the culture temperatures below 35°C during the 3-day culture period. With this harvest frequency (3 days), a 90% harvest of the culture, and avoidance of high temperatures, it was difficult to maintain a harvest biomass level above 60 mg l − 1 dry weight. In the winter, the reduced sunlight and the low ambient temperatures in the greenhouse limited biomass growth. By lengthening the harvest intervals to 4 days a culture biomass level of 30 mg l − 1 dry weight was attainable. Beyond a 4-day culture period minimal additional growth was observed.
Fig. 3. Example of the control chart maintained for the HACH 1720C turbidimeter measuring process of estimating the culture chamber algal biomass.
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Fig. 4. Profile of the growth for CHAET 10 in the algal chambers under greenhouse conditions during the summer and winter months.
3.2. Control operations of algal deli6ery and nursery system There were several concerns with regards to delivering algae from the algae production unit to the seed clam nursery unit. The major concern was the error associated with estimating the alga culture biomass. Underestimating the algal biomass can result in the clams being overfed and inefficient use of the algae produced. An overestimate can result in the underfeeding of the seed clams thus lowering growth rates and potentially increasing the mortality rates. Thus, a cost-effective approach was required for estimating the algal biomass. The low range turbidimeter provided acceptable performance and stability, as indicated by the control chart (Fig. 3), for estimating the algal biomass. Utilizing an in-line fluorimeter (approximately 10× more expensive than the turbidimeter) was not considered cost-effective for the minimal gain in performance (approximately 5%). Utilization of an algal biomass sensor comprised of a photovoltaic solarcell and light source, and much less expensive than the turbidimeter (100× less expensive) was rejected as the unit required constant calibration and performance reliability was inconsistent. Another concern was with the computer-controlled volume exchanges between the algal and seed clam system. The volume of water discharged from the seed clam unit by backflushing the beadfilter or purging the solids separator was replaced by
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delivering an equal volume of algae from the algal system. The computer-controlled volume exchanges were within the control chart UCL and LCL limits and changes to the delivery setup were not necessary (i.e. adjustment of the algal pump activation time period or reservoir purge time period). There was also a concern with the cell integrity and viability from the shear stress in using centrifugal pumps for harvesting, transferring and recirculating the microalgae. Microscope observation indicated cell walls remained intact and no difference in whole cell counts was observed before and after harvesting algae from the culture chambers or after transfer from the algal harvest reservoir to the clam system reservoir. Additionally, when recirculating the algae in the nursery system without seed clams present and bypassing the beadfilter, cell counts and suspended solids analysis did not indicate a reduction in cell numbers or biomass. Another reason for the concern with regards to accurate estimates of algal biomass and volume exchanges was the need to avoid a high algal concentration in the seed clam system. A high algal concentration in a seed clam system can result in pseudofeces production and inefficient algal utilization (Tenore and Dunstan, 1973). Pseudofeces production represents a loss of potentially utilizable algal cells and the production of organic matter in the form of mucous. Consequently, fouling of the seed bed increases which minimizes uniform water flow and food distribution through the seed bed. All of these conditions result in the decrease of seed clam growth. Therefore, a pulsed feeding strategy was utilized to avoid high cell concentrations in the system and minimize psuedofeces production. The transfer of algae from the algal system harvest reservoir to the clam feed reservoir was 2–4 times a day, depending on the alga concentration and availability. The resulting algal concentration in the seed clam system after each feed transfer ranged from 50 000 to 200 000 cells ml − 1. As stated above, before each feeding, a volume of water equal to the volume of algae being transferred was removed from the seed clam system. This volume of water, removed by backflushing the bead filter or by purging the solids separator, resulted in a water volume exchange per feeding from 11 to 40%. The resulting total daily volume exchange ranged from 20 to 160%. With such high daily system volume exchange, incorporating the biofilter into the recirculation process is not necessary and eliminating the biofilter would reduce any algal ration loss due to filter entrapment.
3.3. Culture of seed clams The growth of the seed clams in the integrated system cultured under high-density, fluidized-flow conditions is presented in Fig. 5. The culture trial was terminated after 83 days, due to a sustained decline in the ambient air temperature of the greenhouse resulting in sub-optimal culture unit water temperature for seed clam growth and insufficient cultured algae for a food supply. The mean daily and weekly water quality data are summarized in Table 2. There were no major differences in the progression of the water quality parameters with time during the culture period except for water temperature. The water temperature for the seed
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Fig. 5. Profile of the growth for Mercenaria seed clams utilizing fluidized flow in a computer-controlled integrated algal-seed clam nursery system.
clam system progressively declined due to the seasonal changes in temperature and lack of temperature control in the greenhouse. After 2 weeks of culture from initial stocking, seed survival was 76.8% ( 9 11.5%) with an unexpectedly low growth rate (0.015 per day). Compared to previous laboratory data for forced-flow upweller culture of Mercenaria seed, the growth rate was approximately 70% lower. The stocking density was thus thinned from 3.7 g wet weight clam cm − 2 to 2.0 g wet weight clam cm − 2. After an additional four Table 2 Water quality results of the land-based nursery system utilizing recirculation and fluidized-flow technology Variable
Average
Temperature (°C) 19.0 Dissolved oxygen (mg l−1) 7.2 pH 7.5 Salinity (ppt) 26.0 Ammonia-N (mg l−1) 0.005 Nitrite-N (mg l−1) 0.27 Nitrate-N (mg l−1) 14.1 Total alkalinity (mg l−1 CaCO3) 103.1
Standard deviation
Maximum value Minimum value
1.8 0.6 0.2 1.1 0.017 0.55 5.8 10.3
28.0 9.0 7.9 28.0 0.090 2.30 26.4 121.0
16.6 6.0 7.3 24.0 0.000 0.01 3.8 70.0
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and a half weeks of culture, minimal growth (0.4 mm shell length) of the seed clams was observed and survival rates were lower, 67.3% (9 6.7%). During this 52-day culture period, there was noticeable byssal thread attachment among the smaller clams in the upweller culture units. To minimize and breakup the byssal thread attachment amongst the smaller seed, the actuator valve to the second header tube was closed for 1 min every hour. The resulting increased water flow through each upweller (approximately 25% increase) helped breakup seed clumping, but was not sufficient. It is interesting to note that some of the air which was displaced after the beadfilter was backflushed escaped into the upwellers units instead of through the header tube and was more effective at expanding and breaking up the seed mass, and clearing settled solids from the seed bed. As a result of the byssal thread attachment amongst the smaller seed in the culture units, seed less than 3.0 mm in shell length were removed. The mean shell length of the seed replaced in the upwellers was 3.6 mm (S.D. 9 0.5) and the resulting bed porosity increased from 0.49 to 0.67. A week after the small seed were removed (day 59), the growth rate of the seed improved from 0 to 0.01 per day. On day 73, 3 weeks after the removal of the small seed, the mean shell length increased from 3.6 ( 90.5) to 4.4 ( 90.8) mm. The growth rate tripled to 0.029 per day and survival rate improved to 88.0% (9 1.7%). The shell length measurements indicated a wide distribution of seed size, therefore, a second sorting was conducted to separate the larger seed from the smaller seed. The larger seed, those retained on a 5 mm sieve, were distributed into two upwellers at a density of 3.0 g wet weight cm − 2. The remaining seed, those retained on a 3 mm sieve, were distributed into the remaining four upwellers at the same density. The trial period was terminated 10 days later because sufficient quantities of cultured algae for the seed clams were unavailable and declining ambient temperature inside the greenhouse. During the last 10 days of culture, the average growth rates between the two groups improved to 0.059– 0.063 per day, respectively. Survival rates dropped to 78.1% (9 6.5%) for the units with the smaller seed and 72.0% (9 4.4%) for those with the larger seed. The handling stress from the sorting procedures and lower water temperatures may have potentially resulted in the lower survival rates. Once the smaller seed were removed on day 52 from the culture units, the bed porosity remained steady, ranging from 0.62 to 0.67. The trial results indicating growth rates, bed porosity, shell length, stocking densities, feeding rations and strategies are summarized in Table 1. 4. Discussion The integrated control system performed dependably, sampling and harvesting the alga culture chambers as required, estimating the alga biomass and delivering the calculated volume of algal food within the control limits, and purging the solids separator and backflushing the bead filter as directed. During the trial period there were no valve or pump malfunctions or computer failures resulting in inactivity of control procedures. However, more operating time is needed to evaluate the durability of the system.
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Automating the production of algae provides an interesting alternative to the current manual batch culture method for algae culture at the laboratory and offers the potential to reduce the daily culture labor activities. The majority of the labor savings (approximately 30%) were experienced in reducing the time involved with maintaining and harvesting cultures of different volumes (2 and 10 l carboys, 200 l kalwalls). Furthermore, the algae produced by the automated system were quantified, thus permitting efficient use of the algae when being feed to the seed clams. In addition to employing computer-control to limit labor needs another objective was to provide the Mercenaria seed clams with a dependable and consistent source of algal food for maintaining growth. A ‘crash’ or contamination of the algal culture can result in a loss of a large investment in the animals. Consequently, part of the harvest strategy was to withdraw the maximum amount of usable algae from the system when the desired culture phase was attained. There were constraints that limited consistent and reliable long-term algal production. The primary constraint was operating under ambient greenhouse conditions (variable light and temperature). Other significant culture constraints were bacteria and protozoan contamination, and the biofilm buildup on the tank walls, particularly in the summer. A large harvest volume combined with refilling harvested cultures with treated seawater helped reduced culture contamination and biofilm growth. This batch culture procedure extended culture operation to approximately three weeks before tank cleanings and reinoculation with fresh alga cultures were necessary. The control program was modified to allow the removal of an algal culture tank from system operation for this management process. This was perceived as a more practical method than trying to maintain sterility of large algal cultures on a continual basis, which may not be achievable on a commercial clam operation because of the expense. Fluidization of the seed bed may be a potential culture technique for the high-density land-based nursery culture of Mercenaria seed. Typical seed clam culture at the laboratory involves placing a single layer of seed (initial stocking density approximately 0.2 g wet weight cm − 2) into the upweller culture units and using a low water flow rate to pass cultured algae through the seed mass. By increasing the flow to fluidization velocities in the upwellers, the initial seed stocking density was increased more than 10-fold (3.0 g wet weight cm − 2). However, for smaller seed (B3-mm shell length), the fluidized-flow conditions induced greater byssal thread attachment amongst the seed. The bysall thread attachment caused clumping of the seed mass, thereby affecting water flow and food distribution and reducing uniform seed growth. The clumping of the seed mass was minimized and the seed bed cleaned of settled solids by passing air through the seed mass. Incorporating an air scour of the seed mass versus the hydraulic expansion warrants further evaluation especially if culture conditions involve high densities and fluidization of small size seed. Additionally, a pulse feeding strategy was incorporated to limit the food concentration to the seed, because a high feeding level can result in the production of psuedofeces and inefficient utilization of the feed. When a 4% daily ration was
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provided and divided into four feedings per day, psuedofece production was not observed and over 90% of the algal feed was cleared from suspension within the 6 h feeding time. However, the need for a daily ration greater than the recommended 1.5–2% (Coutteau et al., 1994) appeared to be a result of particle entrapment by the bead filter since as much as 50% of the supplied algal feed was observed to be removed by the bead filter. The daily exchange of the system water volume was high, 40– 160% of the seed clam system volume. This high water exchange volume offsets the need of the bead filter for ammonia removal and reduces alga losses by filter entrapment, which could otherwise be available for the seed. The approximate total cost of the integrated system was $23,100. This cost included the purchase of the personal computer, software, and all components for the water treatment, alga culture, clam culture, and computer-control units. All components of the system described here are readily available and the expense for each system unit was 28.4, 21.5, 25.3, and 24.8%, respectively. With greater availability of alga products (pastes and dried matter) and computer control materials, there are a number of options which commercial facilities may utilized for system control at considerable lower costs. Low-cost programmable logic controllers (about US $100 and up) or single board computers (about US $300 and up) programmed to delivery a premeasured alga paste solution may offer commercial facilities the benefits of optimized, automated feeding system for shellfish without the need of an alga production or water treatment units. The automated feeding of alga paste material warrants further investigation for significant technological advancement to culture systems.
Acknowledgements This work was funded by the University of Georgia Marine Extension Service and the Georgia Sea Grant College Program under grant number NA66RG0282. The Georgia Sea Grant College Program is an element of the National Sea Grant College Program under the direction of National Oceanic and Atmospheric Administration, U.S. Department of Commerce. The authors thank Dr Randal Walker, Dr James Nelson, and Charles Robertson, and other staff at the University of Georgia Shellfish Aquaculture Laboratory and Skidaway Institute of Oceanography for use of facilities and assistance during the system operation and evaluation. The authors also thank Dr Steve Jodis of Armstrong Atlantic State University for his assistance with control program modifications. Mention of a specific product or trade name does not constitute an endorsement nor imply its approval to the exclusion of other suitable products.
References Borland International Inc., 1990. Turbo Pascal User’s Guide, Version 6.0. Scotts Valley, CA 95067-0001
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Castagna, M.A., Kraeuter, J.N., 1977. Mercenaria culture using stone aggregate for predator protection. Proc. Nat. Shellfish Assoc. 67, 1–6. Chen, J.F. 1991. Commercial production of microalgae and rotifers in China. In: W. and K.L. Main (Eds.) Rotifer and Microalgae Culture Systems. Proceedings of a U.S.-Asia Workshop. Fulks. Oceanic Institute, Honolulu, HI, pp. 105–112. Coutteau, P., Hadley, N.H., Manzi, J.J., Sorgeloos, P., 1994. Effect of algal ration and substitution of algae by manipulated yeast diets on the growth of juvenile Mercenaria mercenaria. Aquaculture 120, 135 – 150. Johansen, J.R., Braclay, W.R., Nagle, N., 1990. Physiological variability within ten strains of Chaetoceros muelleri (Bacillariophyceae). J. Phycol. 26, 271 – 278. Montgomery, D.C., 1997. Design and Analysis of Experiments, fourth ed. Wiley, New York. Nelson, J.R., Guardia, S., Cowell, L.E., Heffernan, P.B., 1992. Evaluation of microalgae clones for mass culture in a subtropical greenhouse hatchery: growth rates and biochemical composition at 30°C. Aquaculture 106, 357–377. Tenore, K.R., Dunstan, W.M., 1973. Comparison of feeding and biodeposition of three bivalves at different food levels. Marine Biol. 21, 190–195. Ver, L.M.B., Wang, J.K., 1995. Design criteria of a fluidized bed oyster nursery. Aquac. Eng. 14, 229 – 249. Walker, R.W., Heffernan, P.B., Hurley, D.H., 1997. Growth of Mercenaria mercenaria larvae and juveniles fed two microalgae species under greenhouse conditions in coastal Georgia. Georgia J. Sci. 55, 407 –411. Wikfors, G.H., Ferris, G.E., Smith, B.C., 1992. The relationship between gross biochemical composition of cultured algal foods and growth of the hard clam, Mercenaria mercenaria (L.). Aquaculture 108, 135 – 154.
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