초록

○ 연구결과
ㆍ연안 갯벌 모델로서 여자만 조사 지역의 생․지․화학적 특성 조사
ㆍ연안 갯벌 우수 환경 복원용 미생물 선별
ㆍ연안 갯벌로 유입되는 유기 물질의 미생물학적 제거 모델 개발
ㆍ연안 갯벌 환경복원용 미생물제재의 산업화를 위한 시제품 개발 및 평가

Abstract ▼

Ⅳ.Results of study
The distribution and month changes of the tidalflat macrobenthos community and sedimentary environments was performed on Yeoja Bay (34°47′02,93″,127°34′19,35″) from September 2010 to June 2013. Survey sites were separated into control group without transplanting of P. aibuhite

Ⅳ.Results of study
The distribution and month changes of the tidalflat macrobenthos community and sedimentary environments was performed on Yeoja Bay (34°47′02,93″,127°34′19,35″) from September 2010 to June 2013. Survey sites were separated into control group without transplanting of P. aibuhiteusis, and experiment group with P. aibuhiteusis (100/m²), macrobenthos and sedimentary were sampled and analyzed once a month. The macrobenthos was composed of three phyla and 20 species (control group), and four phyla and 20 species (experiment group). The dominant species of the control and experiment groups was Batillaria multiformis, and subdominant species were Nephtys sp.
There was no P. aibuhiteusis appeared until the 2nd year in the non-transplanting area, and juvenile P. aibuhiteusis was collected in the third year, which could be thought as the effects of transplantation.
In order to investigate the resolving ability of P. aibuhiteusis against pollutants in the coastal tidalflat contaminated with toxic organic substances, endosulfan was selected as the target decomposing compounds. The synthetic wastewater was composed of peptone and glucose.
Laboratory facilities were prepared and experiments were conducted respectively to develop the biological environmental restoration technology for improving the environment of polluted mudflat.
Acute toxicity experiment of endosulfan on the P. aibuhiteusis showed that there was no mortality when the initial concentration was below 10 mg/L, and 26.7% died at 10 mg/L, 48.3% died at 15mg/L, 63.3% died at 20mg/L, respectively in 48h. According to the regression equation, the 24h and 48h LC₅₀ were 55.57 and 15.56 mg/L, respectively.
Results of the acute toxicity of peptone on the P. aibuhiteusis showed that, 48h, 72h, 96h LC₅₀ within 95% confidence limits were 950.35, 340.34, and 120.22 mg/L respectively. The tolerance experiment results of Djadjariensis, Anadara granosa and Manila clam against peptone showed that, there was no mortality in the control group without peptone, and the survival rate decreased with increasing peptone concentration in the experiment groups.
Endosulfan degradation experiments using the P. aibuhiteusis was investigated at 5-day intervals after in jecting 3 ppm endosulfan which based on acute toxicity experiment results. The highest endosulfan sulfate concentration accumulate in the P. aibuhiteusis body appeared on the 15^th day, and then decreased rapidly, while concentrations of endosulfan sulfate accumulated in tidalflat was minor, so we estimated that the endosulfan sulfate was decomposed in the body of P. aibuhiteusis but not discharge.
Decomposition of synthetic wastewater using P. aibuhiteusis showed that, for mesocosm of tidalflat committed to synthetic wastewater, there was no significant difference between control and experiment groups in ignition loss, TOC (total organic carbon), TN (total nitrogen) and TP (total phosphorus), and there was no obvious tendency overtime too. This may be caused that the carbon and nitrogen content in injected 500 ppm synthetic wastewater was too less compared to the tidalflat itself. (about1/10000).
The results of the decomposing of the synthetic wastewater by microorganisms separated from the tidalflat showed that TOC concentration increased to 0.741% from 0.178% on the 4^th day and then decreased to 0.456% on day 10 in control group. At the end of the experiment, TOC concentration in microorganisms A and B increased 0.354% and 0.269%, respectively, did not significantly differ from the initial concentration. Similar trends were also observed in experiment group microorganisms A+B, and group microorganisms A+B+P. aibuhiteusis.
Results of utilizing effective microorganisms and oxidant (oxygen generators) according to the environmental changes showed that, pH and ORP (oxidation reduction potential) increased together in experiment group where microorganism mixed with CaO₂. The reason for this was considered due to the effect of oxygen discharge from the oxygen generation agent like CaO₂.
Infiltrationrate, survival, and growth of P. aibuhiteusis under different sediment particle size showed, 39.7±4.2P. aibuhiteusiss infiltrated, and reached the highest infiltration rate 79.3±8.3% when the particle size was smaller than 0.10 mm. Infiltration rate decreased with increasing particle size. The optimum survival 90.9±4.0% and daily growth rate 0.25% was observed when particle size was smaller than or equal to 0.10 mm.
When sand content decreased and mud content increased, P. aibuhiteusis ingested sand and organic matter together and the composition of sand particles decreased in the rearing tank. X-ray results showed there was particles like sand in the head of P. aibuhiteusis, but not in the middle and tail portions
This study was performed for the purpose of developing a nearshore wetland bioremediation strategy using marine microorganisms. Marine microorganisms were cultured and isolated in marine agar (MA), 1/2MA, 1/10MA media from various sources such as nearshore wetland, polychaete habitats and non-habitats, polychaete, and enrichment culture using organic pesticide.
Microorganisms isolated from nearshore wetland belong to 11 genera, 16 species. which include Algoriphagus halophilus, Halobacillus truperi, Micrococcus luteus, Nautella italica, Photobacterium sp., Pseudoalteromonas byunsanensis, Pseudoalteromonas haloplankti, Pseudoalteromonas lipolytica, Pseudoalteromonas luteoviolacea, Pseudoalteromonas rubra, Pseudomonas sp., Rheinheimera aquimaris, Ruegeria mobilis, Shewanella aquimarina, Vibrio azureus, and Vibrio diabolicus. Among microorganisms belonging to 12 genera from polychaete non-habitats, Bartonella sp., Planomicrobium chinense, Kocuria sp., and Pontibacter sp. were not found in polychaete habitats.
From polychaete habitats, marine organisms which belong to 14 genera, which include Streptomyces sp., Halobacillussp., Sphingopyxis granuli, Lysobacter sp., Marinobacterium rhizophilum, Rhodobacter capsulatus and Idiomarina seosinensis. Thirty five bacterial strains were isolated from polychaete, which were placed into 7 genera such as Bacillus, Micrococcus, Pantoea, Pseudoalteromonas, Rheinheimera, Shewanella, Vibrio.
It is notable that very diverse species of Bacillus were found in polychaete (Perinereis aibuhitensis). Five Bacillus strains, designated CBW3, CBW4, CBW9, CBW14, and EBW10 were characterized and comparatively analyzed, which were capable of aerobic and anaerobic growth as well as degrading various organic compounds.
16S rRNA sequence analysis revealed that strains CBW3 and CBW14 shared 99.8% similarity with that of B. barbaricus, CBW4, CBW9 and EBW10 shared 92.7%, 99.8% and 99.8% similarity with B. anthracis, B. algicoa and B. thuringiensis, respectively. All the bacteria were found to have the enzymes for the degradation of casein and starch. In particular, strain EBW10 had the protease, amylase, cellulase. lipase enzyme activities which could be used toutilize organic macromolecules. Strain EBW4 which had the high capability for the degradation of organic compounds as well as was considered euryhaline novel bacterium had 97.96% similarity in 16S rRNA sequence with B. hemocentroti JSM076093^T. This bacterium was found to have the ability to degrade organic compounds through the experiment using high concentration of synthetic wastewater (10,000 ppm) under euryhaline conditions.
The 1st enrichment culture using organic pesticide end osulfan and plychaete habitat samples resulted in the isolation of six bacterial strains (designated as EDA, EDB, and EDSW) belonging to 5 genera and 6 species, which include Echinicola vietnamensis, Rhodococcus pyridinivorans, Ruegeria mobilis, Thalassobius gelatinovorus, Thalassospira tepidiphila, Tenacibaculum aiptasiae. Among them, strain EDB2 capable of end osulfan degradation was placed into a species of Rhodococcus, and biochemically and physiologically characterized.
Through the second enrichment culture, a microbial consortium consisting of 4 bacteria (Ebht1, Ebht2, Ebht3 and Ebht4) was obtained, which were identified as Rhodococcus ruber, Pseudomonas resinovorans, Pseudomonas putida, Pseudomonas oleovorans. This consortium was revealed a powerful candidate to produce a microbial agent for bioremediation of nearshore wetland owing to the high capability to degrade various organic hydrocarbons including endosulfan. Further study on the degradation ability of the consortium members for organic hydrocarbons revealed that Ebht2 and Ebht4 did not make a great effect on the biodegradation potential in the consortium. Therefore, a consortium composed of Ebht1 and Ebht3 was tested for the degradation of organic compounds using synthetic wastewater made up of glucose and peptone at the ratio 1:4,suggesting the organic compound degradation rate is higher in the consortium than in each bacterium of the consortium.
The consortium was tested with variable components of the media to develop a efficient microbial agent in the environmental industry, resultingin the selection of the best efficient medium component in the aspect of economy and cell growth. The test for activities of the enzymes such as mannase, protease, amylase was performed, which are involved with degradation of various oganic compounds.
Test for the removal of organic compounds by the developed liquid microbial agent under several temperature conditions showed that it removed approximately 70% of 10,000 ppm organic compounds in 12 days under the given conditions. For powder formulation of the consortium microorganisms an excipient for stabilization in nearshore wetland and industrialization was used.
The final product was made after 10% freeze drying microorganisms, 77% yellow clay, 10% glucose and 3% vitamin C were added to the excipient. Test for the removal of organic compounds by the product under several temperature conditions using 100ppm and 1000 ppm of the synthetic wastewater revealed that over 80% of organic compounds was removed very efficiently under25∼30℃. Notably, over10% of organic compounds were degraded even under 5℃ at which most microorganisms lose the physiological and biochemical activities. These result suggested that the formulated microbial agent usefully can be applied in the environmental industry.

목차 Contents

• 표지 ... 1
• 제출문 ... 2
• 요약문 ... 3
• SUMMARY ... 8
• CONTENTS ... 14
• 목 차 ... 16
• List of Tables ... 18
• List of Figures ... 21
• 제1장 연구 개발 과제의 개요 ... 25
• 제2장 국내외 기술개발 현황 ... 26
• 제3장 연구개발 수행내용 및 결과 ... 28
• 제1절 연안갯벌 저서 동물 환경 복원력 평가 ... 28
• 1. 조사지 선정 및 생ㆍ지ㆍ화학적 특성조사 ... 28
• 2. 결과 및 고찰 ... 36
• 제2절 소생태계 실험구 조성 및 갯지렁이 환경복원력 평가 ... 66
• 1. 독성 유기 물질(엔도설판)과 합성 폐수의 선정 ... 66
• 2. Mesocosm system을 이용한 실내 실험 ... 68
• 3. 결과 및 고찰 ... 78
• 제3절 환경복원용 미생물 분리 및 동정 ... 117
• 1. 갯벌,갯지렁이 서식지,갯지렁이 내생 미생물 분리 및 특성 조사 ... 117
• 2. 결과 및 고찰 ... 126
• 제4절 연안 갯벌 환경복원 미생물제재 개발을 위한 효율적인 컨소시엄 구축 ... 165
• 1. 미생물 컨소시엄 후보 선발 ... 165
• 2. 결과 및 고찰 ... 168
• 제5절 미생물 컨소시엄을 이용한 미생물 제재 개발 ... 187
• 1. 미생물 제제 생산 및 분석방법 ... 187
• 2. 미생물 제제 생산을 위한 최적 산업 배지 조건 ... 188
• 3. 결과 및 고찰 ... 192
• 제4장 목표달성도 및 관련분야에의 기여도 ... 199
• 제5장 연구개발 성과 및 성과활용 계획 ... 201
• 1. 논문게재 성과 ... 201
• 2. 특허 성과 ... 203
• 3. 제품 및 시장 분석 ... 206
• 제6장 연구개발과정에서 수집한 해외과학기술정보 ... 205
• 1. 특허분석 ... 205
• 2. 논문분석 ... 205
• 3. 제품 및 시장 분석 ... 206
• 제7장 참고문헌 ... 207
• 부록-유전자원 등록 16S rRNA 염기서열 및 유기물 분해 미생물의 유전자 정보 ... 213
• 연구개발보고서 초록 ... 254
• 끝페이지 ... 254