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1 TR-2011-AQ Report of National Fisheries Research & Development Institute West Sea Fisheries Research Institute, Aquaculture Division 국립수산과학원

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5 Ⅰ. 제목 Ⅱ. 연구개발의목적및필요성 Ⅲ. 연구개발의내용및범위 1

6 Ⅳ. 연구개발결과 2

7 Ⅴ. 연구개발결과의활용계획 3

8 Summary Ⅰ. Title SUPER-INTENSIVE SHRIMP PRODUCTION Ⅱ. Objects Green Growth, we believe is the only option if we are to surmount the difficulties the world now faces due to climate change and economic problems. Korea adheres the Green Growth policy and framework on its way to sustainability as a leading and responsible industrialized country in the world. Biofloc technology (BFT) consists of a variety of bacteria, fungi, microalgae, and other organisms suspended with detritus in culture water. This method significantly decreases the huge amount of aquaculture waste discharge. Its use will impact as decreased coastal eutrophication and mitigated introduction of viral pathogens via water-exchange into the farms that can result to mass mortalities of farmed shrimp. Such will help the shrimp farming industry in its search for truly sustainable management practices. This study demonstrates enhanced production in commercial scale practices for shrimp cultures as well as reduced negative environmental effects in the Yellow Sea. Ⅲ. Development contents Based on the results of primary researches about shrimp culture undertaken last 2003 to 2007, this study developed a commercial scale shrimp culturing method using an advanced new technology from 2008 to Research was carried out as follows (3 patents): 1. Super intensive indoor nursery culture system 2. Super intensive outdoor liner pond culture system 3. Super intensive greenhouse culture system 4. Effect of different concentrations of biofloc on growth and immune activity of shrimps 5. Heterotrophic bacteria species composition of BFT 6. Comparison of nutritional body composition of BFT cultured and pond cultured shrimps Ⅳ. Development results 1. Super intensive nursery culture system in indoor West Sea Fisheries Research Institute (NFRDI) developed specific tank system (3 patents) 4

9 using biofloc technology. Nursery rearing with non-exchange of rearing water according to the biofloc technology, resulted in shrimp harvests twice a year. The heterotrophic bacteria present in rearing water was found able to reduce food conservation rate (FCR) and stimulate growth of shrimps. It was also possible to induce shrimp growth despite high density in indoor culture system. 2. Super intensive lined pond culture system in outdoor From 2008 to 2009, culture methods for two kind of shrimps, F. chinensis and L. vannamei, were tested to find out the possibility of a 2-cropculture per year using HDPE lined outdoor ponds. Results show induced high mortality due to cannibalism when stocking density of F. chinesis is high even though enough food is supplied. Also, L. vannamei was found suitable as the second crop in a 2-crop culture if supply of seedling (postlarvae) is well timed. The growth of Pacific white shrimp in the super intensive, lined pond culture (550 m2 2) was a 19.1g mean body weight, harvest was 1.75 kg/ m2. This productivity is 6 times more than in that of the common pond culture. The mean body weight of shrimps from culture system was 13.2 g and it's productivity was 1.02 kg/ m2. This result is about 3 times more than that of the common pond culture. These results establish the superiority of mixotrophic method to characterize a super intensive lined pond culture. This method, as it minimizes discharge of wastewater contributes to heightened farm biosecurity and encourages recycling of waste material when biofloc is present. 3. Super intensive culture in greenhouse system Two (2) shrimp culture trials were undertaken in commercial scale greenhouse where the biofloc technology effects a biological control that is eco-friendly as well. In the first trial, the density of shrimp was found to be 20 times higher than that of the usual commercial ponds. The productivity in the system with bio-floc was 32.9 times per harvest and harvest may be 65 times in a year. Such productivity figures are equivalent to the production from 3 ha common outdoor pond (water surface area of 600 m 2 ). FCR of this system is about 20 times higher and food consumption was 40~50% lower than traditional pond method. In the second culture trial, production was also 15.9 times higher and the amount of wastewater produced was down by 98%. CO 2 emission was also decreased by 80% due to the heat 5

10 exchange with ground water (38,400 CO 2 ton reductions for the 10 MT shrimp production). 4. Effect of different concentrations of biofloc on growth and immune activity of shrimp The immune activity and growth of shrimp were tested with five different biofloc concentration in rearing water (four treatments at BF100%, BF75%, BF50% and BF25% plus one control (BF0%). In the first trial the growth rate of shrimps in biofloc system increased by 6~27.5% and the activity of the gene propo was also 1.42~3.25 times more. In the second trial the growth rate in biofloc system was significantly higher than control group and PO activity was 3 times more. Result from study in 2009, them RNA expression levels of six immune-related genes were 11~145 times higher in all biofloc groups (BF100, BF75, BF50 and BF25) than in control (BF0). These results strongly suggest that bioflocs give positive effects on growth and immune activity of shrimp. (In 2010, we elucidated 6 immune related genes [propo, PPAE, Ran, Mas, SP1] of cdna sequence from F. chinensisa nd 7genes [propo1, propo2, PPAE, Ran, Mas, SP] from L. vannamei [array no show, only size]). 5. Heterotrophic bacteria species composition of BFT The composition of heterotrophic bacteria in biofloc technology system was analysed using a pyrosequencing method. A total of 13 phyla, 35 orders and 2,000 species were found. The total bacterial counts in shrimp cultures varied from /ml to /ml. 6. Comparison of the body composition of biofloc cultured and pond cultured shrimps. No significant difference was found between animal protein fed and plant protein fed shrimps in comparing the two systems of culturing shrimps. However, shrimps cultured in BFT system showed higher fatty acid content, a deciding factor in the taste of shrimp. Ⅴ. Use of development results Given that the BFT system is not only unique but more importantly, economic and eco-friendly to the environment than recirculation aquaculture system (RAS), development of super intensive culture system incorporating BFT was decided to be composed of 3 projects. First, is education of common shrimp farmers through a Farm Technical Training Center. Second, is the establishment of a standard method for BFT model systems. Third, is the adaptation of BFT system in culturing high value fish species. 6

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23 FIGURE LISTS Fig. 1. Biofloc (BFT) culture system; water quality can be bio-controlled in culture tank. Nitrogenous wastes consume by heterotrophic bacteria and convert to bacterial protein. One part of converted protein is digested by culture animal and the other goes into denitrification process. 26 Fig. 2. Vertical diagram of rearing tank (Patent Nr , 및 ). 30 Fig. 3. Rearing tank (a: panorama of rearing tank, b: water injector c: protein skimmer and NO 3 settling tank). 30 Fig. 4. Water quality change during nursery culture. 33 Fig. 5. Change of weekly growth rate of white Pacific white shrimp during nursery culture. 34 Fig. 6. Circular concrete tank for intermediate grow out culture (a : rearing tank, b : equipment, protein skimmer, venturi recirculation pump, NO3 settling tank etc.). 36 Fig. 7. Change of water quality indicators in the intermediate grow out trials. 39 Fig. 8. Weekly growth rate of white leg shrimp during intermediate grow out period. 41 Fig. 9. Outdoor HDPE lined grow out pond(a) and panorama(b). 43 Fig. 10. Daily changes of water quality indicators for outdoor HDPE-lined ponds during grow out period. 46 Fig. 11. Change of weekly growth rate of Pacific white shrimp in outdoor HDPE lined ponds. 48 Fig. 12. Weekly growth rate of Pacific white shrimp in Pond 1 and Fig. 13. Weekly body weight change of Pacific white shrimp in Pond 1 and Fig. 14. Change of water quality in Pond 1 and Fig. 15. Change of water quality in rearing water of outdoor HDPE lined pond (pond 1and 2). 55 Fig. 16. Rearing tank of biofloc technology. 56 Fig. 17. Ground plan of greenhouse culture system using biofloc technology. 57 Fig. 18. Side view of greenhouse culture system using biofloc technology. 57 Fig. 19. Change of cumulative weekly body weight of Pacific white shrimp in Tank 1 and Fig. 20. Weekly growth rate of Pacific white shrimp in Tank 1 and Fig. 21. Body weight distribution of Pacific white shrimp in Tank 1 and Fig. 22. Change of water quality in rearing water of Tank 1 and 2 (from 7th Apr. to 18. Aug.). 61 Fig. 23. Change of water quality in rearing water of Pacific white shrimp (1st trial; ). 64 Fig. 24. Change of water quality in rearing water of Pacific white shrimp during greenhouse culture (2nd trial; ). 65 Fig. 25. Change of total heterotropic bacteria in outdoor HDPE lined pond during nursery culture of shrimp. 68 Fig. 26. Change of Vibrio counts during shrimp culture in outdoor HDPE lined pond)

24 Fig. 27. Variation of total bacteria count in rearing water of Tank 1 and Fig. 28. Comparison of total bacteria and Vibrio spp. counts in Tank Fig. 29. Total bacteria and Vibrio spp. number in rearing water of Tank Fig. 30. (left) early stage of rearing water. DAPI fluorescent stain ( 1,000); (middle) Biofloc developed rearing water; (right) Biofloc well developed rearing water. Bacteria aggregated with organic material and built the biofloc. 72 Fig. 31. Total bacterial counts in greenhouse rearing tank 1 and Fig. 32. Total bacterial counts in outdoor rearing pond A and B. 74 Fig. 33. Pyrosequencing result of biofloc rearing water- species richness under phylum level (in number). 75 Fig. 34. Pyrosequencing result of biofloc rearing water- species richness under phylum level (in percentage). 76 Fig. 35. Pyrosequencing result of biofloc rearing water- species richness under order level (in percentage). 76 Fig. 36. Pyrosequencing result of biofloc rearing water- species richness under (refraction). 77 Fig. 37. Cloning process of immune genes. 79 Fig. 38. Total bacterial counts with different biofloc concentrations. 80 Fig. 39. Hemocyte in phase-contrast light microscopy. GC: granular cell; HC: hyaline cell; SGC: semigranular cell. 82 Fig. 40. Relative comparison of mrna expression of immune related 6 genes in Pacific white shrimp larvae. 84 Fig. 41. Protein concentration in Pacific white shrimp hemocyte with different biofloc concentration. 85 Fig. 42. Phenoloxidase (PO) activity in hemocyte of shrimp with different biofloc ratio. 85 Fig. 43. Total bacteria counts in nursery culture of shrimp with different biofloc concentrations (DAPI fluorescent stain, X1,000). 88 Fig. 44. Relative rate of hemocyte type in hemolymph of shrimps reared in different biofloc concentration, GC : granular cell, SG: semigranular cell, and HC: hyaline cell. 89 Fig. 45. Body weight and survival rate of Finneropenaeus chinensis cultured with different biofloc concentration. 91 Fig. 46. Total bacteria counts with different biofloc concentration. 91 Fig. 47. Change of Vibrio counts with different biofloc concentration. 92 Fig. 48. Body length and weight of adult Fenneropenaeus chinensis cultured with different biofloc concentration. 94 Fig. 49. PO activity of adult Fenneropenaeus chinensis cultured with different biofloc concentrations. 94 Fig. 50. Total bacteria counts in rearing water of different biofloc concentration (DAPI fluorescence stain, x1,000) 95 20

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27 Table List Table 1. Comparison of biofloc technology and common outdoor pond culture system 28 Table 2. Larvae input into rearing tanks for nursery culture 31 Table 3. Variation of water quality and nutrient concentration in no water exchange nursery culture using BFT(mean and range) 32 Table 4. Production and survival rate of shrimps during nursery culture with limited water exchange 35 Table 5. Juvenile stocking and intermediate grow-out trials 37 Table 6. Change of water quality indicators and nutrient concentration in intermediate grow out trials of white leg shrimps using BFT (mean and range) 38 Table 7. Intermediate grow out trials of white leg shrimp under limited water exchange 40 Table 8. Outdoor shrimp stocking into HDPE-lined grow out pond 44 Table 9. Changes of water quality indicators and nutrient concentrations in HDPE lined grow out ponds using Biofloc technology 45 Table 10. Summary of stocking and production of Pacific white shrimp in HDPE-lined ponds using heterotrophic bacteria 47 Table 11. Summary of Pacific white shrimp production (high density of postlarva and juvenile) in outdoor rearing ponds 49 Table 12. Summary of water quality indicators in rearing water of Pond 1 and 2 (mean, range) 51 Table 13. Results of Pacific white shrimp production in outdoor HDPE lined pond under limited water exchange condition 53 Table 14. Water quality indicators during outdoor HDPE lined pond culture (mean value) 54 Table 15. Production and survival rate of Pacific white shrimp during grow out culture using biofloc technology ( ~8. 31) 58 Table 16. Change of water quality of Pacific white shrimp grow out culture using biofloc under limited water exchange 61 Table 17. Result of Pacific white shrimp in 1st( ) and 2nd trials ( ) 62 Table 18. The mean values of water quality during Pacific white shrimp culture in 1st ( ) and 2nd ( ) trials 63 Table 19. Composition of heterotrophic bacterial community in nursery culture with non-water exchange system (102 days rearing) 66 Table 20. Comparison of total heterotropic bacteria and Vibrio spp. in nursery culture tanks with non-water exchange system 67 Table 21. The percent of Vibrio spp. to heterotrophic bacteria in rearing water of outdoor HDPE 23

28 lined pond during grow out period 69 Table 22. Total bacterial and vibrio spp. counts in greenhouse rearing tank 1, 2 and outdoor pond 1, 2 during grow out period 74 Table 23. Shrimp growth rate and FCR with different biofloc concentration (in triplicate) 79 Table 24. Initial total bacteria count with different biofloc concentration in rearing water 80 Table 25. Summary of water quality with different biofloc content (mean, range) 81 Table 26. Growth and survival rate of Pacific white shrimp with different biofloc 86 Table 27. Mean values of water quality with different biofloc concentration 87 Table 28. Comparison of mrna expression of immune related 6 genes of Pacific white shrimp larvae reared under different biofloc concentrations 89 Table 29. Change of body weight of postlarva Fenneropenaeus chinensis with different biofloc concentrations 90 Table 30. Mean value of water quality with different biofloc concentrations 92 Table 31. Growth of adult Fenneropenaeus chinensis with different biofloc concentrations 93 Table 32. Nutritional composition of shrimp cultured from different feeds (animal protein and plant protein). A; animal protein source (pond 1, RW 1), B; plant protein source (pond 2, RW 2) 96 Table 33. Nutritional composition of Pacific white shrimp, cultured from different feeds (animal protein and plant protein). A; animal protein source (pond 1, RW 1), B; plant protein source (pond 2, RW 2) 96 Table 34. Fatty acid composition of shrimp and feed. A; animal protein source (pond 1, RW 1), B; plant protein source (pond 2, RW 2) 97 24

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32 표 1. 기존축제식새우양식과 BFT 를이용한신기술의비교 Table 1. Comparison of biofloc technology and common outdoor pond culture system 28

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34 그림. 2. 사육수조및시설측면모식도 (2007 년특허등록번호 , 및 ). Fig. 2. Vertical diagram of rearing tank (Patent Nr , 및 ). (a) (b) (c) 그림 3 사육수조사진 (a: 사육수조전경, b: 저면분사장치, c: 포말분리기및탈질산조 ). Fig. 3. Rearing tank (a: panorama of rearing tank, b: water injector, c: protein skimmer and NO 3 settling tank). μ 30

35 표 2. 중간육성시험구와종묘입식현황 Table 2. Larvae input into rearing tanks for nursery culture Rearing tank Surface area(m 2 ) Total number of shrimp Nursery culture Density (Shrimp/ m2 ) Biomas (g/ m2 ) Mean B.W.(g) Raceway , Raceway , Raceway , Raceway , C, 27.5 C 31

36 표 3. 사육수비교환방식 (BFT) 을적용한중간육성시험구의사육수질및영양염의농도변화 ( 평균및범위 ) Table 3. Variation of water quality and nutrient concentration in no water exchange nursery culture using BFT(mean and range) Rearing tank Water temp.(c) DO ( mg/l) Salinity ph TAN ( mg/l) NO 2 -N ( mg/l) Alkalinity ( mg/l) Turbidity (NTU) molasses (L/day) RW RW RW RW

37 그림 4 중간육성시험구의사육기간중수질변화. Fig. 4. Water quality change during nursery culture. 33

38 그림 5. 흰다리새우중간육성시험구별주간성장률변화. Fig. 5. Change of weekly growth rate of white Pacific white shrimp during nursery culture. 34

39 표 4. 사육수비교환방식을이용한새우중간육성실험의생산량및생존율 Table 4. Production and survival rate of shrimps during nursery culture with limited water exchange Raceway tank Initial B.W.(g) Stocking density Yield Days Final B.W.(g) (/ m2 ) (/ m3 ) (kg/ m2 ) (kg/ m3 ) Estimated Sur. rate(%) Estimated FCR RW RW RW RW

40 φ 그림 6. 원형콘크리트중간양성수조사진 (a : 사육수조전경, b : 시설장비류, 포말분리스키머, 벤츄리순환펌프, 탈질산조등 ). Fig. 6. Circular concrete tank for intermediate grow out culture (a : rearing tank, b : equipment, protein skimmer, venturi recirculation pump, NO 3 settling tank etc.). 36

41 표 5. 중간양성시험구와종묘입식 Table 5. Juvenile stocking and intermediate grow-out trials Intermediate grow-out Rearing tank Surface area(m 2 ) Total number of shrimp Density (Shrimp/ m2 ) Biomas (g/ m2 ) Mean B.W.(g) RT , RT ,

42 표 6. 사육수비교환방식 (BFT) 을적용한흰다리새우중간양성실험구의사육수수질환경및영양염농도변화 ( 평균및범위 ) Table 6. Change of water quality indicators and nutrient concentration in intermediate grow out trials of white leg shrimps using BFT (mean and range) Rearing tank Water temp.( ) DO (mg/l) Salinity ph TAN (mg/l) NO2-N (mg/l) Alkalinity (mg/l) Turbidity (NTU) molasse s(l/day) RT RT

43 7. 중간양성시험구의사육기간중수질변화. Fig. 7. Change of water quality indicators in the intermediate grow out trials. 39

44 표 7. 사육수비교환방식을적용한흰다리새우중간양성시험구의사육결과 Table 7. Intermediate grow out trials of white leg shrimp under limited water exchange Rearing tank Area (m 2 ) Initial B.W.(g) Stocking density Yield Final B.W.(g) ( m2 ) (/ m3 ) (kg/ m2 ) (kg/ m3 ) Estimated Sur. rate(%) Estimatd FCR RT RT

45 그림. 8. 중간양성기간중흰다리새우의주간성장률변화. Fig. 8. Weekly growth rate of white leg shrimp during intermediate grow out period. 41

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47 그림 9. 야외포장사육지의평면모식도 (a) 및사진 (b). Fig. 9. Outdoor HDPE lined grow out pond(a) and panorama(b). 43

48 g/ m3, 흰다리새우 0.35 g/ m3였다. 표 8. 야외포장사육지양성시험종묘입식 Table 8. Outdoor shrimp stocking into HDPE-lined grow out pond Rearing pond Species Working volume( m3 ) Grow-out (HDPE-lined pond) Total number of shrimp Density (Shrimp/ m3 ) Biomas (g/ m3 ) Mean B.W.(g) LP 1 L. vannamei , LP 2 F. chinensis ,

49 표 9. 사육수비교환방식 (BFT) 을적용한포장사육지양성시험구의사육수수질환경및영양염농도변화 ( 평균및범위 ). Table 9. Changes of water quality indicators and nutrient concentrations in HDPE lined grow out ponds using Biofloc technology Rearing pond Water temp.( ) DO ( ) Salinity ph TAN ( ) NO 2 -N ( ) Alkalinity ( ) Turbidity (NTU) Molasses (L/day) LP LP

50 그림 10. 야외포장사육지양성시험구의사육기간중수질변화. Fig. 10. Daily changes of water quality indicators for outdoor HDPE-lined ponds during grow out period. 46

51 표 10. HDPE-lined pond 에서의타가영양세균을이용한새우양성결과요약 Table 10. Summary of stocking and production of Pacific white shrimp in HDPE-lined ponds using heterotrophic bacteria Rearing pond B.W. (g) Stocking Total no. of shrimp Density (shrimp/ m3 ) Culture period (days) B.W. (g) Harvest Total yield (kg) production (kg/ m3 ) survival (%) FCR LP , LP ,

52 그림 11. HDPE-lined 야외사육지에서의새우의주간성장률변화. Fig. 11. Change of weekly growth rate of Pacific white shrimp in outdoor HDPE lined ponds. 나. 2차고밀도야외사육지양성시험 48

53 표 11. Postlarva 와중간육성종묘의야외사육지고밀도양식결과요약 Table 11. Summary of Pacific white shrimp production (high density of postlarva and juvenile) in outdoor rearing ponds No. stocked PL Stocking density(/m 2 ) Initial B.W.(g) Days Final B.W.(g) Total production (kg) Yield (kg/m 2 ) Survival rate(%) FCR Pond 1 102, Pond 2 102,

54 그림 12. Pond 1 과 2 에서의흰다리새우주간성장률비교 Fig. 12. Weekly growth rate of Pacific white shrimp in Pond 1 and 2 그림 13. Pond 1 과 2 에서의흰다리새우누적주간체중변화비교. Fig. 13. Change of cumulative weekly body weight of Pacific white shrimp in Pond 1 and 2. 50

55 표 12. Pond 1 과 2 의사육수수질환경변화요약 (mean, range) Table 12. Summary of water quality indicators in rearing water of Pond 1 and 2 (mean, range) Water Temp.(C) DO (mg/l) Salinity (mg/l) ph TAN (mg/l) NO 2-N (mg/l) NO 3-N (mg/l) Alkalinity (mg/l) Turbidity (NTU) molasses (L/day) Pond Pond

56 그림 14. Pond 1 과 2 의수질환경변화. Fig. 14. Change of water quality in Pond 1 and 2. 52

57 다. 3차고밀도야외사육지양성시험 1, 표 13. 사육수비교환에의한야외사육지고밀도양식결과 Table 13. Results of Pacific white shrimp production in outdoor HDPE lined pond under limited water exchange condition Pond Pond

58 표 14. 사육기간중야외사육지수질환경요인의평균값 Table 14. Water quality indicators during outdoor HDPE lined pond culture (mean value) W.T. ( ) DO (mg/l) Salinity ph TAN (mg/l) NO 2 -N (mg/l) NO 3 -N (mg/l) Chl-a (ug/l) TSS (mg/l) VSS (mg/l) Alkalinity (mg/l) Turbidity (NTU) pond 1 pond

59 그림 15. HDPE 야외사육지 pond 1 과 pond 2 의수질환경요인의변화. Fig. 15. Change of water quality in rearing water of outdoor HDPE lined pond(pond 1and 2). 55

60 그림 16. BFT 방식의사육조모형도. Fig. 16. Rearing tank of biofloc technology. 56

61 그림. 17. 그린하우스 BFT 양식장의평면도. Fig. 17. Ground plan of greenhaus culture system using biofloc technology. 그림 18. 그린하우스 BFT 양식장의측면도. Fig. 18. Side view of greenhaus culture system using biofloc technology. 57

62 표 15. 사육수비교환미생물총기술 (BFT) 를이용한흰다리새우본양성의생산량및생존율 ( ~8. 31) Table 15. Production and survival rate of Pacific white shrimp during grow out culture using biofloc technology ( ~8. 31) Initial B.W.(g) Stocking density(/m 2 ) Days Final B.W.(g) Yield (kg/m 2 ) Total production (kg) Survival rate(%) FCR Tank Tank

63 그림. 19. Tank 1 과 2 의새우의누적주간체중변화. Fig. 19. Change of cumulative weekly body weight of Pacific white shrimp in Tank 1 and 2. 그림 20. Tank 1 과 2 의흰다리새우의주간성장율비교. Fig. 20. Weekly growth rate of Pacific white shrimp in Tank 1 and 2. 59

64 그림 21. Tank 1 과 2 의새우의체중분포. Fig. 21. Body weight distribution of Pacific white shrimp in Tank 1 and 2. 60

65 표 16. 사육수비교환미생물총기술 (BFT) 를이용한흰다리새우본양성의사육수수질변화 Table 16. Change of water quality of Pacific white shrimp grow out culture using biofloc under limited water exchange W.T. ( ) DO (mg/l) Salinity ph TAN (mg/l) NO 2-N (mg/l) NO 3-N (mg/l) Chl-a ( μg /L) TSS (mg/l) VSS (mg/l) Alkalinity (mg/l) Turbidity (NTU) Tank Tank 그림 22. Tank 1 과 2 의수질환경변화 ( 월 ~ 8 월 ). Fig. 22. Change of water quality in rearing water of Tank 1 and 2 (from 7th Apr. to 18. Aug.). 61

66 표 차 ( ) 및 2 차 ( ) 흰다리새우사육실험결과 Table 17. Result of Pacific white shrimp in 1st ( ) and 2nd trials ( ) 1st trial 2nd trial initial BW (g) stocking density (/m 2 ) period (Day) final BW (g) production (kg/m 2 ) total production (kg) survival (%) FCR Tank Tank Tank Tank Mean ,

67 μ μ 표 차 ( ) 및 2 차 ( ) 사육시수질환경요인의평균값 Table 18. The mean values of water quality during Pacific white shrimp culture in 1st ( ) and 2nd ( ) trials W.T. ( ) DO (mg/l) Salinity ph TAN (mg/l) NO 2-N (mg/l) NO 3-N (mg/l) Chl-a (ug/l) TSS (mg/l) VSS (mg/l) Alkalinity (mg/l) Turbidity (NTU) 1st trial 2nd trial tank 1 tank 2 tank 1 tank

68 그림 23. 그린하우스흰다리새우사육수내수질요인의변화 1 차 ( ). Fig. 23. Change of water quality in rearing water of Pacific white shrimp (1st trial; ). 64

69 그림. 24. 그린하우스흰다리새우사육수내수질요인변화 (2 차 ; ). Fig. 24. Change of water quality in rearing water of Pacific white shrimp during greenhouse culture (2nd trial; ~ ). 65

70 표 19. 사육수비교환중간양성수조에서의타가영양세균군집 (hetero-trophic bacterial community) 의종조성 ( 사육 102일째 ) Table 19. Composition of heterotrophic bacterial community in nursery culture with non-water exchange system (102 days rearing) Heterotrophic bacteria Community ratios (%) RT 1 (Round tank 1) RT 2 (Round tank 2) Tennacbaculam sp Fluorecent bacteria Microbacterium sp Roseobactor sp White colonies Other groups

71 4.78 E + 06 CFU, RT 2 에서는 8.07 E + 05 CFU로 RT 1에서타가영양세균의수가많았다. 비브리오균수는 RT 1에서 4.10 E + 02 CFU, RT 2에서 6.00E + 02 CFU로 RT 1보다 RT 2에서다소높게나타났다. 비브리오균수에대한타가영양세균수가 RT 1에서더높게나타났다. 사육수내수질분석결과 ( 표 6) 과비교하면 RT 1에서의탁도가 NTU로 NTU인 RT 2보다다소높았는데이는사육수내조성된타가영양세균의군집에의한것으로파악되었으며흰다리새우또한 RT 1에서의체중이더높아타가영양세균에의하여성장이촉진되는것으로파악되었다. 표 20. 사육수비교환중간양성수조에서의타가영양세균및비브리오세균비교 ( 사육 102일째 ) Table 20. Comparison of total heterotropic bacteria and Vibrio spp. in nursery culture tanks with non-water exchange system Heterotrophic bacteria 평균세균수 (CFU/ ml ) RT 1 (Round tank 1) RT 2 (Round tank 2) Total heterotrophic bacterial count 4.78 E E + 05 Vibrio spp. count 4.10 E E + 02 Total bac. count / Vibrio count (%) 1.17 E E

72 그림. 25. 야외포장사육지양성기간동안총타가영양세균수의변화 Fig. 25. Change of total heterotropic bacteria in outdoor HDPE lined pond during nursery culture of shrimp. 68

73 그림 26. 야외포장사육지양성기간동안총비브리오균수변화. Fig. 26. Change of Vibrio counts during shrimp culture in outdoor HDPE lined pond. 표 21. 야외포장사육지양성기간중사육수에서조사된총 heterotrophic bacteria 수에대한 Vibrio 균수의비율 (%) 변화 Table 21. The percent of Vibrio spp. to heterotrophic bacteria in rearing water of outdoor HDPE lined pond during grow out period Days LP LP

74 4 ( 그림 27. Tank 1 과 2 의총세균수의변화. Fig. 27. Variation of total bacteria count in rearing water of Tank 1 and 2. 70

75 그림 28. Tank 1 의총세균수와비브리오수의비교. Fig. 28. Comparison of total bacteria and Vibrio spp. counts in Tank 1. 그림 29. Tank 2 사육수의총세균수와비브리오수의비교. Fig. 29. Total bacteria and Vibrio spp. number in rearing watger of Tank 2. 71

76 그림 30. ( 좌 ) 사육초기의사육수. DAPI 형광염색현미경사진 ( 1,000); ( 중앙 ) Biofloc 발달중인사육수 ; ( 우 ) Biofloc이잘발달된사육수. 세균이유기물과결합하여 biofloc을형성한다. Fig. 30. (left) early stage of rearing water. DAPI fluorescent stain ( 1,000); (middle) Biofloc developed rearing water; (right) Biofloc well developed rearing water. Bacteria aggregated with organic material and built the biofloc. 라. 비교환사육수 (biofloc) 의세균학적특성조사 μ 72

77 그림 31. 그린하우스 tank 1, 2 의총세균수 Fig. 31. Total bacterial counts in greenhouse rearing tank 1 and 2. 73

78 그림 32 야외사육지 pond A, B 의총세균수. Fig. 32. Total bacterial counts in outdoor rearing pond A and B. 표 22. 사육기간동안그린하우스의 tank 1, 2 및야외사육지 pond 1, 2의총세균수및비브리오수의평균값 Table 22. Tatal bacterial and vibrio spp. counts in greenhouse rearing tank 1, 2 and outdoor pond 1, 2 during grow out period Total bacterial counts Vibrio counts tank 1 tank 2 pond 1 pond 2 tank 1 tank 2 pond 1 pond 2 MIN 1.2E E E E+06 MIN 6.6E E E E+02 MAX 8.7E E E E+07 MAX 4.6E E E E+03 S.D. 2.2E E E E+06 S.D. 1.3E E E E+03 MEAN 5.3E E E E+06 MEAN 2.3E E E E+03 74

79 그림 33. 기간에따른 biofloc 사육수의 pyrosequencing 결과-phylum level에서의세균의다양성 ( 세균수 ). Fig. 33. Pyrosequencing result of biofloc rearing water- species richness under phylum level (in number). 75

80 그림 34. 기간에따른 biofloc 사육수의 pyrosequencing 결과 -Phylum level 에서의세균의다양성 ( 비율 ). Fig. 34. Pyrosequencing result of biofloc rearing water- species richness under phylum level (in percentage). 그림 35. 기간에따른 biofloc 사육수의 pyrosequencing 결과 -order level 에서의세균의다양성. Fig. 35. Pyrosequencing result of biofloc rearing water- species richness under order level (in percentage). 76

81 그림 36. 기간에따른 biofloc 사육수의 pyrosequencing 결과 - species richness (refraction). Fig. 36. Pyrosequencing result of biofloc rearing water- species richness under (refraction). 77

82 78

83 표 23. Biofloc 농도별실험구의성장률과사료효율 (3 반복실험 ) Table 23. Shrimp growth rate and FCR with different biofloc concentration (in triplicate) Exp I Exp II Exp III Exp IV Control Initial B.W.(g) Final B.W.(g) Total biomass (g) Consumed feed (g) FCR Survival rate (%) % biofloc Exp. biofloc. 100% biofloc TAN NO 2 biofloc. 그림. 37. Biofloc 농도별실험구의새우성장률. Fig. 37. Shrimp growth rate with different biofloc concentrations. 79

84 Exp. groups EXP I EXP II EXP III EXP IV Control Mean 그림 38. Biofloc 농도별실험구의총세균수. 각 회계수. Fig. 38. Total bacterial counts with different biofloc concentrations. TSS VSS biofloc. TAN NO 2 Exp. I 0.59, 2.0 ppm biofloc. 80

85 표 25. Biofloc 농도별실험구의수질환경요약 (mean, range) Table 25. Summary of water quality with different biofloc content (mean, range) EXP Ⅰ EXP Ⅱ EXP Ⅲ EXP Ⅳ Control Water Temp.( ) DO (mg/l) Salinity ph TSS (mg/l) VSS (mg/l) TAN (mg/l) NO 2-N (mg/l) NO 3-N (mg/l) Chl.-a (ug/l) μ μ μ 81

86 그림 39. 위상차현미경에의한헤모사이트의사진, GC: granular cell; HC: hyaline cell; SGC: semigranular cell. Fig. 39. Hemocyte in phase-contrast light microscopy. GC: granular cell; HC: hyaline cell; SGC: semigranular cell. μ μ μ μ 82

87 환산계수 cell 수 (cells/ml) = A x (F.A O.A) Filtered sample 량 (ml) A, counts of bacteria; F.A., filtered area; O.A., observed area 83

88 그림 40. Biofloc 농도별사육수에사육된새우의혈림프내 hemocyte type 별상대비율. GC : granular cell, SG: semigranular cell, and HC: hyaline cell. Fig. 40. Relative rate of hemocyte type in hemolymph of shrimps reared in different biofloc concnetration, GC : granular cell, SG: semigranular cell, and HC: hyaline cell. 84

89 그림 41. Biofloc 농도별사육된흰다리새우 hemocyte 의단백질농도의변화. Fig. 41. Protein concentration in Pacific white shrimp hemocyte with different biofloc concentration. 그림 42. Biofloc 농도에따른새우사육시 의페놀옥시다제 (PO) 활성도.. 85

90 표 26. 서로다른농도의 biofloc에서사육된흰다리새우의성장률및생존율 Table 26. Growth and survival rate of Pacific white shrimp with different biofloc BF0 BF25 BF50 BF75 BF100 BF0-SV BF100-SV B.W.(mg) a a,b a,b a,b b c c S.D min max Survival (%) 83 a 89 a 85 a 92 a 96 a 2 c 33 b (p<0.05) 86

91 μ 표 27. Biofloc 농도에따른시험구별수질요인의평균값 (3일간격측정 ) Table 27. Mean values of water quality with different biofloc concentration Parameter BF0 BF25 BF50 BF75 BF100 p WT( ) 27.8 a 27.8 a 27.8 a 27.6 a 27.5 a Salinity( ) 32.3 a 33.9 a 33.6 a 33.4 a 33.3 a ph 8.6 a 8.4 b 8.2 c 8.1 c 7.7 d 0.00 D.O(mg/L) 5.8 a 5.0 b 4.9 b 4.8 b 4.9 b 0.00 Chl-α(μg/L) a a a,b b b TA-N(mg/L) a b 0.62 c c c 0.00 NO 2 -N(mg/L) 0.01 a 0.12 a 0.53 a,b 0.77 a,b 1.12 b NO 3 -N(mg/L) 0.28 a b b b b 0.00 TSS(mg/L) 66.3 a b c d e 0.00 VSS(mg/L) a b c d d 0.00 (p<0.05) 87

92 그림 43. 각실험구의총세균수 (DAPI 형광염색, X1,000 에서계수 ) 비교 Fig. 43. Total bacteria counts in nursery culture of shrimp with different biofloc concentrations (DAPI fluorescent stain, 1,000) 88

93 표 28. 서로다른 biofloc 농도에서사육된흰다리새우유생의 6개면역관련유전자의 mrna expression 상대적비교 Table 28. Comparison of mrna expression of immune related 6 genes of Pacific white shrimp larvae reared under different biofloc concentrations propo propo2 PPAE mas sp1 ran BF BF BF BF BF 그림 44. 흰다리새우유생 (postlarva) 의 6 개면역관련유전자의 mrna expression 상대적비교. Fig. 44. Relative comparison of mrna expression of immune related 6 genes in Pacific white shrimp larvae. 89

94 표 29. 다양한농도 biofloc 사육수에서사육된대하 PL 의체중변화 Table 29. Change of body weight of postlarva Fenneropenaeus chinensis with different biofloc concentrations BF0 BF25 BF50 BF75 BF100 BF0-SV BF100-SV Survival(%) 52 a 36 b 36 b 30 b 32 b 1 c 1 c Mean BW a a,b,c a,b a,b a c b,c B.W.min B.W.max SD

95 그림 45. biofloc 에사육된대하 PL 의체중및생존율. Fig. 45. Body weight and survival rate of postlarva Fenneropenaeus chinensis cultured with different biofloc concentrations. 그림 46. 기간별각 biofloc 시험구의총세균수 (DAPI 형광염색, x1,000). Fig. 46. Total bacteria counts with different biofloc concentrations. 91

96 그림 47. 기간별각 biofloc 시험구의 Vibrio 수. Fig. 47. Change of Vibrio counts with different biofloc concentrations. 표 30. 다양한농도의 biofloc 사육수의수질항목평균값 Table 30. Mean value of water quality with different biofloc concentrations Parameter BF0 BF25 BF50 BF75 BF100 P-value (<0.05) Temperature ( ) 28.8 a 28.8 a 28.7 a 28.9 a 28.8 a Salinity( ) 31.7 a 31.4 a,b 30.9 a,b 30.5 b,c 29.9 c ph 8.7 a 8.3 b 8.1 b 8.0 d 7.8 c D.O (mg/l) 5.7 a 5.6 b 5.5 b 5.4 b 5.5b b TAN (mg/l) 0.02 a 0.30 b 0.43 c 0.46 c 0.66 d NO 2 -N(mg/L) a 0.51 a 1.44 b 1.33 b 1.84 b NO 3 -N(mg/L) 0.09 a a a 874 b 1150 c TSS (mg/l) 41.8 a b c d e VSS (mg/l) 5.8 a b c d 556 e

97 표 31. biofloc 사육수에서사육된대하성체의성장 Table 31. Growth of adult Fenneropenaeus chinensis with different biofloc concentrations 93

98 그림 48 대하성체에서의 biofloc 농도별체장과 (B.L) 과체중 ( B.W) 의차이. Fig. 48. Body length and weight of adult Fenneropenaeus chinensis cultured with different biofloc concentrations. 94

99 마. Biofloc 유기새우및축제식양식새우체성분비교 95

100 표 32. 새우및사료의일반성분분석. 실험사료 A, 동물성단백질원 (pond 1, RW 1 공급 ); 실험사료 B, 식물성단백질원 (pond 2, RW 2 공급 ). ( 수분포함기준 ) Table 32. Nutritional composition of shrimp cultured from different feeds (animal protein and plant protein). A; animal protein source (pond 1, RW 1), B; plant protein source (pond 2, RW 2) (%) (%) (%) (%) Pond ± ± ± ±0.04 Pond ± ± ± ±0.01 RW ± ± ± ±0.06 RW ± ± ± ± ± ± ± ±0.01 Pond ± ± ± ±0.01 Pond ± ± ± ±0.01 RW ± ± ± ±0.01 RW ± ± ± ± ± ± ± ± ± ± ± ±0.09 A 9.26± ± ± ±0.07 B 11.00± ± ± ±0.04 표 33. 흰다리새우의일반성분분석. 실험사료 A, 동물성단백질원 Pond 1과 RW1은동물성단백질사료공급 ; pond 2와 RW 2는식물성단백질사료공급 (DM 기준 ) Table 33. Nutritional composition of Pacific white shrimp, cultured from different feeds (animal protein and plant protein). A; animal protein source (pond 1, RW 1), B; plant protein source (pond 2, RW 2) 96

101 표 34. 새우및사료의지방산분석. 사료 A, 동물성단백질원 (pond 1); 사료 B, 식물성단백질원 (pond 2, RW 2 공급 ) Table 34. Fatty acid composition of shrimp and feed. A; animal protein source (pond 1, RW 1), B; plant protein source (pond 2, RW 2) 시료명 사료 일반양식장 Pond-1 Pond-2 RW-1 RW-2 분석항목 일반사료사료A 사료B 가식전어가식전어부체부체 가식부 전어체 가식부 전어체 가식부 전어체 Myristic acid C 14: Myristoleic acid C 14: Pentadecanoic acid C 15: cis-10-pentadecanoic acid C 15: Palmitic acid C 16: Palmitoleic acid C 16: Magaric acid C 17: Magaroleic acid C 17: Stearic acid C18: Oleic, Elaidic acid C 18:1n9c,1n9t Linoleic acid C 18:2n9c γ-linolenic acid C 18:3n,6,9,12c Linolenic acid C 18:3n9,12,15c Arachidic acid C 20: Eicosenoic acid C 20: Eicosadienoic acid C 20: cis-11,14,17-eicosatrienoic acid C 20: Heneicosanoic acid C 21: Arachidonic acid C 20: EPA C 20: Behenic acid C 22: Erucic acid C 22: cis-13,16-docosadienoic acid C 22: Tricosanoic acid C 23: Lignoceric acid C 24: Nervonic acid C 24:1n15c DHA C 22: 6n total

102 제 4 장목표달성도및관련분야에의기여도 제 5 장연구개발결과의활용계획 98

103 제 6 장참고문헌 99

104 100

105 101