[논문]복합미생물 프로바이오틱을 이용한 환경친화적 넙치 순환여과양식시스템에서의 미생물군집 분석

이채영; 김하함; 알핀이마뉴엘; 김홍기; 원성훈; 배진호; 배승철; 고성철

doi:10.7845/kjm.2018.8085

복합미생물 프로바이오틱을 이용한 환경친화적 넙치 순환여과양식시스템에서의 미생물군집 분석
Microbial community analysis of an eco-friendly recirculating aquaculture system for olive flounder (Paralichthys olivaceus) using complex microbial probiotics 원문보기

Korean journal of microbiology = 미생물학회지, v.54 no.4, 2018년, pp.369 - 378

이채영 (한국해양대학교 환경공학과) , 김하함 (국립부경대학교 해양바이오신소재학과) , 알핀이마뉴엘 (한국해양대학교 환경공학과) , 김홍기 ((주)바요) , 원성훈 (국립부경대학교 해양바이오신소재학과) , 배진호 (국립부경대학교 해양바이오신소재학과) , 배승철 (국립부경대학교 해양바이오신소재학과) , 고성철 (한국해양대학교 환경공학과)

초록
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본 연구는 순환여과양식시스템(RAS)에 있어서 복합프로바이오틱스의 적용이 넙치의 성장과 병저항성에 미치는 영향과 이 프로바이오틱스를 RAS에 생물증강처리 시 미생물군집 구조 및 수질에 미치는 영향을 평가하고자 실시하였다. RAS 내에서 80미의 넙치치어($25.7{\pm}7.6g$; $15.2{\pm}1.7cm$)에 프로바이오틱스 CES-AQ1를 첨가하여 사료를 제조하여(CES 사료; $1{\times}10^9\;CFU/kg$) 8주일 동안 급이하였다. 이 경우 넙치의 증체율, 비성장속도, 사료효율, 및 단백질 전환효율은 비유수식 양식시스템에 있어서 CON, PI 및 OTC 사료를 처리한 경우에 비해 1.5~2.5배 정도 높게 나타났다. 1주일간 병원균 저항성 시험에 있어서 비유수식에서 항생제함유 사료(OTC)를 급이한 경우와 RAS에서 CES 사료를 처리한 경우간에는 별 차이가 나타나지 않았다. 따라서 이 CES 프로바이오틱스를 RAS에서 넙치를 양식하는데 있어서 항생제 대용으로 활용할 수 있을 것으로 판단되었다. RAS의 생물여과막에서는 가장 높은 미생물다양성이 나타났으며 암모니아의 산화 및 탈질능을 가진 미생물이 관찰되었고, 병원미생물의 성장억제도 관찰되었다. 더구나 RAS 운전 19일 경과 시 암모니아가 0.5 mg/L이하의 농도로 감소하여 양호한 RAS 수질의 유지에 있어서 프로바이오틱스 처리가 효과가 있음이 밝혀졌다. 사료에 프로바이오틱스(CES-AQ1)를 첨가하여 넙치 장내 미생물이 안정화되고 또한 이 프로바이오틱스를 RAS 양식수에도 처리하여 RAS를 운전할 경우 건강한 넙치의 양식과 양호한 수질을 유지할 수 있어서 경제적이고 환경친화적인 넙치양식이 가능할 것으로 판단되었다.

Abstract ▼ AI-Helper

This study was conducted to evaluate effects of dietary microbial probiotics on the growth and disease resistance of olive flounder (Paralichthys olivaceus) in a recirculating aquaculture system (RAS), and the effects of the probiotic bioaugmentation on the microbial community structure and water quality. For the analysis, 80 juvenile fish (average weight, $25.7{\pm}7.6g$; average length, $15.2{\pm}1.7cm$) were fed a basal diet containing a commercial microbial product CES-AQ1 (CES; $1{\times}10^9\;CFU/kg$ diet) in an RAS for 8 weeks. Weight gain, the specific growth rate, feed efficiency, and protein efficiency ratio of the fish fed the CES diet in the RAS were 1.5~2.5 times higher than those of fish fed the basal diet alone, or the basal diet containing oxytetracycline (OTC), yeast plus bacterium, or Bacillus subtilis in a still water system. There was no significant difference in the pathogen challenge test between fish fed the OTC diet and fish fed the CES diet in the RAS, suggesting the CES-AQ1 probiotic used in the RAS as a potential replacement for antibiotics. The RAS biofilter maintained the highest microbial diversity and appeared to harbor microbial communities with ammonium oxidation, denitrification, and fish pathogen suppression functions. Ammonia, which is hazardous to fish, was significantly decreased to < 0.5 mg/L in 19 days, indicating the effectiveness of probiotic supplementation to maintain good water quality in RAS. These results suggest that the intestinal microbial communities of fish are stabilized by a probiotic-containing diet (CES) and that bioaugmentation with probiotics may be an eco-friendly and economical supplement for aquaculture of olive flounder, promoting both good water quality and fish health in an RAS.

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표/그림 (7)

그림 Fig. 1. Schematic of the recirculating aquaculture system (RAS) used in this study.
그림 Fig. 2. Chemical analysis of water quality in terms of chemical oxygen demand (COD), total nitrogen (T-N), ammonia (NH₃-N), Nitrate (NO₃^--N), and total phosphorus (T-P) in the RAS.
표 Table 1. Growth performance of juvenile olive flounder fed experimental diets and grown in a recirculating aquaculture system (RAS) or still water tanks for 8 weeks
그림 Fig. 3. Cumulative survival of juvenile olive flounder after challenge with Edwardsiella tarda for 7 days.
표 Table 2. Diversity indices of the microbial communities in samples from the water and biofilter of the RAS and the guts of fish grown in the RAS based on pyrosequencing analysis^a
그림 Fig. 4. Microbial community structures in the gut of the fish, water, and biofilter in the RAS at the (A) phylum and (B) species levels.
$Fig. 5. Unifrac UPGMA clustering analysis of the microbial community structures in the guts of fish fed various experiment diets (OTC, Pure isolates, and CES-AQ1) and the guts of fish, as well as the water and biofilter, in the RAS (RAS).$ 그림 Fig. 5. Unifrac UPGMA clustering analysis of the microbial community structures in the guts of fish fed various experiment diets (OTC, Pure isolates, and CES-AQ1) and the guts of fish, as well as the water and biofilter, in the RAS (RAS).

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제안 방법

At the end of the feeding trial, the total number and weight of the fish in the RAS tank were measured to calculate weight gain (WG), feed efficiency (FE), the specific growth rate (SGR), protein efficiency ratio (PER), and survival rate. In addition, three fish were randomly selected, and their hepatosomatic index (HSI, %), viscerosomatic index (VSI, %), and condition factor (CF) were determined.
In this study, an RAS was used to test the possible application of probiotics for olive flounder culture to achieve higher diet efficiency and maintain good water quality. The microbial community was analyzed to understand the effects of dietary probiotics on growth and disease resistance in olive flounder and to address roles of probiotic bioaugmentation in an ecofriendly RAS.
In this study, we evaluated the effects of dietary probiotics on growth and disease resistance in olive flounder (Paralichthys olivaceus) as well as the effect of probiotic bioaugmentation on water quality in an eco-friendly RAS during olive flounder aquaculture. WG, the SGR, FE, and the PER of fish fed the CES diet in the RAS were much higher than those of fish fed the CON, PI, and OTC diets in still water systems.
Then, library construction, sequencing, and sequencing analyses were performed using a 454 GS FLX Junior Sequencing System (Roche), according to the manufacturer’s protocols.
Three other diets were formulated, each of which contained one the following three cultures at 1 × 109 CFU/kg of basal diet: Bacillus subtilis (BS, positive control; Lee et al., 2017), the commercial microbial product CES-AQ1 (CES), and a mixture of the yeast Groenewaldozyma salmanticensis and the bacterium Gluconacetobacter liquefaciens (PI; pure isolates), discovered in this study.

대상 데이터

For the experiment, 80 juvenile olive flounder (initial average weight and length, 25.7 ± 7.6 g and 15.2 ± 1.7 cm, respectively) were introduced into the RAS tank (working volume, 500 L).
The feeding trial was carried out in an aquarium building at Korea Maritime and Ocean University (Busan, South Korea). Olive flounder were obtained from Uljin Aquaculture (South Korea).
5℃ under a 12-h light/dark period during the experiment. The fish pathogen Edwardsiella tarda (ATCC 15947), was obtained from the Department of Biotechnology, Pukyong National University (Busan, Republic of Korea). The bacterial strain originated from a diseased brook flounder.

이론/모형

1. Schematic of the recirculating aquaculture system (RAS) used in this study.

성능/효과

At the end of the feeding trial, the total number and weight of the fish in the RAS tank were measured to calculate weight gain (WG), feed efficiency (FE), the specific growth rate (SGR), protein efficiency ratio (PER), and survival rate. In addition, three fish were randomly selected, and their hepatosomatic index (HSI, %), viscerosomatic index (VSI, %), and condition factor (CF) were determined. These parameters were calculated by using the following equations: weight gain (WG, %) = (final weight - initial weight) × 100/initial weight; specific growth rate (SGR, %/day) = (log_e final weight - log_e initial weight) × 100/days; feed efficiency (FE, %) = (wet weight gain/dry feed intake) × 100; protein efficiency ratio (PER) = (wet weight gain/protein intake); survival rate (SR, %) = (total no.
WG, the SGR, FE, and the PER of fish fed the CES diet in the RAS were much higher than those of fish fed the CON, PI, and OTC diets in still water systems. Moreover, the 7-day challenge test showed there was no significant difference between fish fed with diets containing OTC, PI (in still water systems), or CES (in the RAS) in terms of cumulative survival rate, indicating that PI and CES could be potential antibiotic replacements for disease control in olive flounder. Moreover, an 8-week feeding trial showed that WG, SGR, FE, and PER were 1.
97 mg/L) was observed in 16 days and was maintained at 1~3 mg/L. The concentrations of NO₃^--N, NH₃-N, and T-P were 1~3 mg/L during the first 13 days of the experiment, and then significantly decreased to less than 0.5 mg/L at 19 days, during which all other water quality parameters were stably maintained. In fact, the average ratios of COD/T-N, NH₃/T-N, and NO₃^-/T-N on days 19~64 were 2.
The overall microbial α-diversity indices of the samples from the RAS were several times higher than those of samples from the still water systems, indicating that the growth environment of the fish could significantly affect the microbial diversity in the RAS as well as the fish gut.
During the challenge test, the first death occurred on the first day post-injection. The survival rate of the fish fed the OTC diet and challenged with E. tarda was significantly higher than the survival rate of fish fed the CON, BS, and CES diets. In contrast, there was no significant difference among fish fed the PI and OTC diets for up to 7 days (Fig.
Moreover, there were no significant differences in growth performance between fish fed the CES and OTC diets. This study demonstrated that the dietary administration of CES exerted a beneficial effect on growth performance that was equivalent to that of B. subtilis or antibiotics.

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용어 설명: PCR은 세균 특이성이 있는 primer를 이용하여 적은 수의 세균이 있을지라도 쉽게 검출할 수 있는 유용한 방법이며, 이를 이용하여 구강 내 치면세균막이나 타액에서 직접 세균을 검출할 수 있게 되었다[8].

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