22 DECEMBER • WORLD AQUACULTURE • WWW.WAS.ORG eliminated the need for artificial filtration, allowing the native microbial community, including beneficial bacteriophages, to remain intact. The absence of mechanical filtration in Batch 2 post-transition was intentional, as it preserved microbial richness and allowed for the development of a more ecologically integrated environment. This setup was intended to simulate offshore rearing conditions in a contained setting, providing a realistic model for studying the microbial interactions that may contribute to oyster resilience, immune response, and biomineralization. By allowing microbial connectivity and natural biogeochemical processes to unfold, the Batch 2 system provided a more holistic perspective on sustainable aquaculture practices and the potential benefits of bio-integrated environments in pearl oyster farming. Environmental Variables In order to evaluate the impact of different rearing conditions on oyster health and pearl development, several key environmental parameters were either deliberately varied or closely monitored across the two experimental batches. Water temperature was one of the primary variables. In Batch 1, precise thermal control was achieved using chillers, which maintained the water temperature consistently within a narrow range of 20 to 22°C. This allowed researchers to assess oyster performance under stable, low-stress conditions. In contrast, Batch 2 was exposed to ambient seawater temperatures, which fluctuated naturally between 19 and 24°C. This setup provided insights into the oysters’ physiological responses to moderate thermal variability under more natural conditions. Feeding regimes also differed over time. Initially, both batches received carefully cultured microalgae, as outlined in Section 2.2. However, as the experiment progressed, Batch 2 transitioned to relying entirely on naturally occurring phytoplankton present in the circulated seawater. This shift allowed for an evaluation of the oysters’ nutritional adaptability and the practicality of using natural algal sources in large-scale, low-cost operations. Water exchange strategies were another important variable. In Batch 1, water was partially replaced at scheduled intervals to maintain quality and manage waste buildup. Batch 2, by comparison, benefited from a continuous flow-through system using fresh natural seawater. This approach not only maintained water clarity and oxygenation but also supported a dynamic and self-regulating ecological environment. A crucial distinction between the two systems lay in their microbial exposure. In Batch 1, the water was mechanically filtered, significantly reducing microbial diversity and the presence of native bacteriophages. This created a highly controlled, though somewhat ecologically simplified, environment. In contrast, Batch 2 used unfiltered seawater, which preserved the native microbial community, including potentially beneficial phages. This microbial richness may have contributed to enhanced immune stimulation and overall resilience among the oysters. Together, these environmental variables provided a comprehensive framework for assessing how different levels of ecological control and natural integration influence the biological and commercial outcomes of onshore pearl oyster culture. A detailed comparison of system parameters between Batch 1 and Batch 2 is provided in Table 1. Algal Feed and Water Parameters To ensure optimal nutritional support during the critical phases of post-operative recovery and pearl sac development, three species of live microalgae were employed as feed: Nannochloropsis salina, Chaetoceros calcitrans and Isochrysis galbana. Batch 1 Algae for Batch 1 were cultivated in Walne medium - a nutrientrich formulation containing essential nitrates, phosphates, and trace elements necessary for optimal algal growth. Algal concentrations were maintained at approximately 50,000 cells/mL and administered continuously through a calibrated drip-feeding system tailored to match the oysters’ natural filtration rates. Water temperature was stringently regulated using chillers to maintain a stable range of 20 - 22°C. Batch 2 Initially, during the post-operative care phase, Batch 2 oysters were fed algae cultured under the same Walne-based protocol used for Batch 1. However, following the initial healing phase, Batch 2 was transitioned to a semi-natural system utilizing unfiltered seawater containing ambient microalgae. This setup was designed to mimic offshore rearing conditions and was maintained under ambient light to encourage in situ photosynthesis. Importantly, the absence of artificial filtration preserved native bacteriophages and beneficial microbes, supporting the hypothesis that microbial intelligence plays a vital role in enhancing post-surgical recovery, immune stabilization, and biomineralization. Throughout the experimental period, water quality parameters were carefully monitored and consistently maintained within optimal ranges to support the health and development of the pearl oysters in both batches. Maintaining a stable pH was essential, and values were kept between 8.2 and 8.5. When necessary, sodium bicarbonate was added to buffer the water and ensure that pH levels remained within this biologically favorable window. Salinity was another critical factor, held steady at 31 to 32 parts per thousand (ppt) to closely replicate the oysters’ natural marine habitat. This salinity range supports optimal physiological function in both Pinctada margaritifera and Pteria penguin, ensuring that osmotic balance and metabolic activity were not disrupted during the rearing process. Temperature management varied between the two batches, allowing for a controlled comparison. In Batch 1, temperature was regulated precisely using industrial-grade chillers to maintain a narrow range between 20 and 22°C. This precise thermal control minimized fluctuations and supported a stable internal environment for the oysters, reducing metabolic stress and promoting consistent immune responses. Batch 2, by contrast, utilized continuous natural seawater circulation without artificial temperature control. As a result, water temperature in Batch 2 varied between 19 and 24°C, offering a more naturally modulated environment that served as a control group for evaluating the effects of ambient conditions. Biological filtration systems were employed in both setups but played a particularly vital role in the closed-loop system of Batch 1. Operated for 18 hours each day, these filters were instrumental in breaking down metabolic waste products, particularly ammonia and nitrites, and in maintaining stable nitrogen cycling. This was essential
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