Isolation, cultivation and cell-viability maintenance of useful bacteria suitable for treatment of aquaculture wastewater

Abstract

As a source of healthy protein, seafood is a favorite food for many people, and its demand has increased every year. As a result, it is difficult for the catch to meet this demand, and the production of aquatic products through aquaculture is greatly increasing, and the importance of the aquaculture industry as the 4th industry responsible for future food is drawing attention. However, the development of the aquaculture industry has led to an increase in consumption of aquatic food, and consequently, an increase in the amount of waste and wastewater, resulting in the need for efficient waste/wastewater treatment to preserve the environment and increase production of aquaculture products. For efficient and environmentally friendly aquaculture waste/wastewater treatment, many wastewater treatment technologies using microorganisms have been developed. Microorganisms living in the viscera of rockworms and photosynthetic bacteria are known to be very useful microorganisms in treating wastewater discharged from seawater farm. Therefore, in this study, useful microorganisms suitable for aquaculture wastewater treatment are isolated, mass culture and simple culture technology increase the ease of wastewater treatment of these microorganisms, and microbial stability maintenance technology is used to treat wastewater from actual shrimp farms. In order to increase the efficiency, the possibility of practical use of aquaculture wastewater treatment using useful microorganisms was studied. In Chapter 1, useful microorganisms were isolated from the viscera of the rockworm(Marphysa sanguinea), which inhabits the bottom of the shrimp farm and has the ability to purify sediment, and investigated the wastewater treatment ability of the microorganisms. Samples obtained from the viscera of rockworms were cultured in 6 different nutrient media, and a total of 87 isolates with different characteristics were isolated through colony shape and microscopic observation. Among these isolates, isolates with excellent growth and protein and lipid degradation. After searching, 8 isolates without mutual antagonism were finally obtained. In order to match the characteristics of farm wastewater containing various components, the finally obtained 8 isolates were designed as 12 different microbial combinations with various compositions and then applied to similar farm wastewater. As a result, the microbial combination with excellent protein degrading ability and high viable cell number(4.2 × 106 CFU/mL) was a combination of 6 isolates including Bacillus paramycoides, B. pumilus, Stenotrophomonas sp., 2 B. paranthracis, and Micrococcus luteus. As a result of wastewater treatment of shrimp farming using this complex microorganism, COD and TN were removed by 51.0% and 44.6%, respectively, after 12 hours of treatment, and the C/N ratio was maintained almost constant at 10, so that this complex microorganism group was stably, it has been shown that wastewater can be treated. Therefore, it was confirmed that useful microorganisms isolated from rockworm intestines are suitable for wastewater treatment of shrimp farm. In Chapter 2, photosynthetic bacteria, which have been reported as useful microorganisms that have excellent water purification ability and play an excellent role in bioremediation in the agricultural and livestock sector, are high value-added microorganisms, but are difficult to cultivate simply due to the difficulty of pure culture, reducing their utilization. Therefore, since there is a great need for development and dissemination of development technology related to this, in this study, a culture method that can be used more easily for field input and treatment efficiency of easily cultured photosynthetic bacteria in actual shrimp farm wastewater treatment were investigated. Among the photosynthetic bacteria used, the most suitable strain for scale-up production was Rhodobacter azotoformans EBN-7, which showed the highest specific growth rate (0.20/h) in Basic medium. R. azotoformans EBN-7 strain showed 4.50 × 1010 CFU/mL, dry cell weight of 26.8 g/L, and specific growth rate (0.15/h) in a 500 L pilot-scale culture. As a result of culturing in a 15 L simple incubator using the final culture medium obtained here as a seed, it was confirmed through microscopic observation that photosynthetic bacteria dominate after 3 days of culture. The maximum permissible storage period of R. azotoformans EBN-7 for the production of active seed for on-site wastewater treatment was investigated as 3 months at 4 ℃. When R. azotoformans EBN-7 cultured in a 250 L simple bioreactor was applied to shrimp farm wastewater, NH4+-N was significantly reduced to 60.7% compared to the control group. Therefore, it was found that the R. azotoformans EBN-7 strain cultured by a simple culture technique can be effectively used in shrimp farm wastewater treatment. In Chapter 3, methods to maintain the microbial stability for a long time when useful microorganisms are introduced into the field for wastewater treatment were investigated. The useful microorganisms used in this study were photosynthetic bacteria, and the microbial stability of the photosynthetic bacteria was maintained for a long time using cell immobilisation and lyophilisation technology, and the wastewater treatment efficiency was investigated through field application. Among the divalent cation solutions used for photosynthetic bacteria immobilisation, 0.1 M CaCl2 is the most effective divalent cation solution in terms of no environmental pollution, substrate diffusivity, size (4 mm) favorable for cell loading, and cost effectiveness. Beads prepared using this have a relatively high viable cell number(1.91 × 109 CFU/mL). As a storage method for the photosynthetic bacteria bead produced, the highest microbial stability was shown in a 3.5% NaCl solution at 4 ℃. In the lyophilisation method, the highest microbial stability was confirmed when skim milk (9%) and glucose (2%) were added as cryoprotective additives. When photosynthetic bacteria beads for aquaculture bottom improvement and lyophilisation powder for aquaculture water treatment manufactured under optimal conditions were applied to shrimp farms for 18 weeks, 55% of NH4+ was removed compared to the control group, and NO3- and NO2- 100% removed. Therefore, cell immobilisation and lyophilisation under optimal conditions were able to maintain the microbial stability of photosynthetic bacteria longer, and as a result, it was shown that effective in situ shrimp aquaculture water treatment was possible. Through the studies in Chapters 1-3 above, it was confirmed that the appropriate complex microbial combination of microorganisms living in the viscera of rockworms can be usefully used for wastewater treatment in seawater farms, and the spawning of photosynthetic bacteria difficult to culture through simple culture technology. The production method made it easier to use photosynthetic bacteria when inputting them to the farm wastewater treatment site. It was confirmed that it is very useful for in situ farm water treatment. Therefore, from the results obtained in this study, the importance of developing and commercializing useful microorganisms and related technologies in sustainable aquaculture was confirmed.