Intensive Rotifer Production in a Pilot-Scale Continuous Culture Recirculating System Using Nonviable Microalgae and an Ammonia Neutralizer
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JOURNAL OF THE Vol. 39, No. 5 WORLD AQUACULTURE SOCIETY October, 2008 Intensive Rotifer Production in a Pilot-scale Continuous Culture Recirculating System Using Nonviable Microalgae and an Ammonia Neutralizer C. D. BENTLEY,P.M.CARROLL, AND W. O. WATANABE1,2 Center for Marine Science, University of North Carolina Wilmington, Wilmington, North Carolina 28403-5927 USA A. M. RIEDEL Aquatic Eco-Systems, Apopka, Florida 32703 USA Abstract A study was conducted to test the performance of a high-density (.3000 individuals/mL) continuous recirculating system for rotifers (B. rotundiformis) fed nonviable Nannochloropsis oculata and using sodium hydroxymethanesulfonate to neutralize ammonia. Three different microalgae feed rates (g of N. oculata [68 3 109 cells/mL] per million rotifers/d) were tested in successive trials. In Trial 1 (feed rate 5 1.5), during a 30-d period, rotifers were harvested daily to 3000 individuals/mL, for an average yield of 178 million/d. Feed efficiency (million rotifers/g/d) was 0.33. In Trial 2 (feed rate 5 1.1), during a 32-d period, an average of 106 million rotifers were harvested daily, and feed efficiency was 0.26. In Trial 3 (feed rate 5 1.3), during a 30-d period, an average of 107 million rotifers was harvested daily, and feed efficiency was 0.23. An economic analysis based on a feed rate of 1.5 showed that production cost was 40% lower than the traditional batch culture method (US$ 0.29 vs. 0.46 per million rotifers/d). The continuous culture system tested reliably produced large quantities of rotifers on a daily basis without the use of a biofilter and with a lower production cost than a batch culture system. One of the factors limiting seed production in space requirements and labor, eliminating the a marine finfish hatchery operation is the produc- need for multiple tanks, and improve culture sta- tion of the rotifers Brachionus rotundiformis and bility by stabilizing the bacterial populations in B. plicatilis as a food source for first feeding lar- the culture (James and Abu-Rezeq 1989; Fu et al. vae. It has been estimated that 400 billion rotifers 1997; Yoshimura et al. 1997; Rombaut et al. are required to produce 10 million gilthead seab- 2001). Recently, a recirculating system for continu- ream, Sparus aurata, fry in Mediterranean hatch- ous high-density rotifer culture was described eries (Zmora et al. 1991 cited by Lubzens et al. (Suantika et al. 2000, 2003). This system not only 2001). Many marine finfish hatcheries rely on reduced water consumption but also replaced the traditional ‘‘batch culture’’ method for rotifer microalgae with a cost-effective formulated diet. production in which several tanks are maintained For an intensive culture of rotifers to operate and harvested at timed intervals to ensure a con- efficiently, there must be an adequate supply of tinual supply. This method is labor intensive, re- food to maintain the rotifers in a satiated condi- quires a large portion of a hatchery’s floor space, tion (Hirayama et al. 1973). The development of and is often unstable and unpredictable (Dhert commercially available concentrated microal- et al. 2001). High-density continuous rotifer gae has allowed the culturist to provide the roti- culture systems are currently being developed fers with sufficient amounts of feed without the worldwide to provide daily harvests from one need to produce live microalgae in the hatchery tank. These systems, utilizing microalgae as feed, (Lubzens et al. 1995). This has greatly reduced have been shown to significantly reduce hatchery the need to allocate time and space to live feed production; however, the use of concentrated 1 Corresponding author. microalgae in high-density rotifer culture still 2 Present address: 601 S. College Road, Wilmington, NC represents 86% of production costs (Fu et al. 28403. 1997). Ó Copyright by the World Aquaculture Society 2008 625 626 BENTLEY ET AL. Increasing levels of NH3 within the culture (Dierckens 2005). It is important, therefore, to medium, associated with rotifer population reduce labor associated with rotifer culture sys- growth, have a negative effect on the reproductive tems and to simplify system components to min- rate of both B. rotundiformis and B. plicatilis (Yu imize the potential for human error. and Hirayama 1986; Araujo et al. 2001). One The objective of this study was to evaluate the method for reducing NH3 levels in a culture is performance of a simplified recirculating sys- to regulate the pH at 7.0 through the addition of tem using condensed, nonviable microalgae hydrochloric acid using an automated pH control- Nannochloropsis oculata and using sodium hy- ler (Yoshimura et al. 1996). This method keeps droxymethanesulfonate to neutralize ammonia. the majority of total ammonia nitrogen (TAN) + as nontoxic NH4 and has been used to success- Methods fully culture rotifers at densities from 10,000 to This study was conducted from February to 30,000 individuals/mL. Alternatively , a special- August 2004 at the University of North Carolina ized micropore membrane filtration device has Wilmington Center for Marine Science (UNCW- been used to remove water containing NH3 from CMS) Aquaculture Facility, Wrightsville Beach, the culture without removing algal cells or roti- North Carolina, USA. The UNCW-CMS stock 5 fers, allowing ultrahigh densities (1.6 3 10 culture of B. rotundiformis was used for this study. individuals/mL) to be maintained for 4 d in The same procedures were used for all three trials. a flow-through culture system (Yoshimura et al. 2003). Although not practical in a commercial Culture System operation, this system showed that extremely high The rotifer culture system was situated in an densities are possible if ammonia is removed. A indoor, controlled environment laboratory. The preconditioned biological filter has also been used system consisted of a semisquare, rounded cor- to remove ammonia in intensive rotifer culture to ner 190-L culture tank with a sloping bottom maintain cultures at 3000 individuals/mL for and containing 125-L of culture water (Fig. 1). 34 d (Suantika et al. 2000). A new method to reduce ammonia in rotifer culture involves adding sodium hydroxymethanesulfonate to the culture water which neutralizes unionized ammonia and its toxic effect. This product was effective in decreasing TAN and NH3 in a small-scale rotifer batch culture system (Riche and Pfeiffer 2006). To date, no studies have demonstrated the suc- cessful application of sodium hydroxymethane- sulfonate in batch culture at a practical scale or in continuous, high-density culture systems. The unpredictability of rotifer cultures is a sig- nificant problem among many hatcheries and has been attributed to degrading water quality and unstable bacterial populations within the culture (Dhert et al. 2001; Rombaut et al. 2001). Contin- uous culture systems have improved the stability of rotifer cultures (Fu et al. 1997; Suantika et al. 2000; Dhert et al. 2001; Hagiwara et al. 2001; Rombaut et al. 2001). However, in a recent sur- vey of 13 hatcheries in Europe, where rotifer cul- ture ranging from batch culture to recirculating systems are used, human error was suspected to FIGURE 1. Intensive system with 50-L sump on left, 150-L be the most prevalent reason for culture failures culture tank in middle, and feed cooler on right. ROTIFER PRODUCTION IN A PILOT-SCALE CONTINUOUS CULTURE RECIRCULATING SYSTEM 627 Seawater was pumped from a natural source Aeration was provided to the culture tank adjacent to the lab. Culture water was prepared from a blower through four 4-cm air diffusers, by mixing 1-mm filtered, UV-sterilized seawater and pure oxygen was delivered from a dewar with municipal freshwater to achieve a salinity through a 15-cm microdiffuser. Temperature was of 20 ppt. For further sterilization, the culture controlled by a 300-W glass submersible heater medium was chlorinated for 24 h with liquid placed in the culture tank and was maintained bleach solution (6.5% ClONa) and then dech- at 25.0–29.9 C. Natural lighting was provided lorinated with sodium thiosulfate. through a laboratory window, and fluorescent The culture tank was fitted with a center stand- overhead lights were used only during system pipe drain consisting of a 10-cm-diameter plastic maintenance. netting frame covered with a 55-mm nylon mesh screen to retain the rotifers in the culture vessel Feeding and an inner standpipe to maintain the culture To begin a rotifer culture trial, the system was volume (Fig. 2). A bubble ring at the bottom of initially stocked at a density of 600–900 indivi- the standpipe decreased fouling of the mesh. duals/mL and the culture density was monitored Twenty-six liters per hour (500%) of system daily. Nonviable microalgal paste N. oculata water was recirculated through the culture tank (approximately 68 3 109 cells/mL) (Reed each day. Water exiting the culture tank drained Mariculture, Campbell, CA, USA) was used to by gravity into a 50-L sump tank, and water feed the rotifer culture. Approximately 10 g was then circulated by a 1.5-amp pump through was added directly to the culture tank, and the a foam fractionator at a rate of 24.2 L/min. to re- remainder of the algae paste was diluted in move dissolved and suspended solids. A bypass 16 L of 20 ppt water and stored in a cooler with diverted the remainder of the flow into the sump. ice packs. The diluted algal paste was added The total system volume was 220 L (Fig. 3). continuously to the culture tank over a 22-h FIGURE 2. Center drain for culture unit with 55-mm screen. Four filter mats are suspended from center drain. 628 BENTLEY ET AL. FIGURE 3.