초록 ▼

1. 국내 자생 목재미생물의 기초 특성 규명
- 국내 자생 목재미생물의 균류학적 특성(유전적 특성, 형태학적 특성, 생육특성) 평가
- 국내 자생 목재미생물의 생리적 특성(유기산 생성 능력, 균체외효소 생성 능력) 평가
- Mycoremediation용 우수균주의 특이 유전자 탐색
2. 국내 자생 목재미생물의 mycoremediation 특성 규명
- 국내 자생 목재미생물의 유해오염물질(중금속, PAHs, 합성염료) 저항 및 제거 특성 평가
- 목재미생물을 이용한 실제 환경 내 오염물질(중금속 오염폐

1. 국내 자생 목재미생물의 기초 특성 규명
- 국내 자생 목재미생물의 균류학적 특성(유전적 특성, 형태학적 특성, 생육특성) 평가
- 국내 자생 목재미생물의 생리적 특성(유기산 생성 능력, 균체외효소 생성 능력) 평가
- Mycoremediation용 우수균주의 특이 유전자 탐색
2. 국내 자생 목재미생물의 mycoremediation 특성 규명
- 국내 자생 목재미생물의 유해오염물질(중금속, PAHs, 합성염료) 저항 및 제거 특성 평가
- 목재미생물을 이용한 실제 환경 내 오염물질(중금속 오염폐수, 중금속 오염토양, PAHs 오염토양, 염색폐수) 제거 기술 개발
3. 환경생물자원 종합정보시스템 구축
- 위키기반 유용환경생물자원 정보시스템의 개발
- 환경복원용 균류의 특성확인을 위한 분자 기능 분류 기법의 개발 및 적용
- 유전체정보를 활용한 균류 유전자 클러스터 예측 및 균류의 분류기법 개발들로부터의 피드백을 통한 시스템의 유지보수

Abstract ▼

This project was carried out to evaluate basic characteristics and mycoremediation characteristics of wood-inhabiting fungi and to construct the integrated environmental bioresource information system. The research was partitioned into a series of substudies: (1) Investigation of basic characteristi

This project was carried out to evaluate basic characteristics and mycoremediation characteristics of wood-inhabiting fungi and to construct the integrated environmental bioresource information system. The research was partitioned into a series of substudies: (1) Investigation of basic characteristics of wood rot fungi inhabiting in Korea; (2) Investigation of mycoremediation characteristics of the wood rot fungi; (3) Construction of the integrated environmental bioresource information system.
A total of 309 isolates of wood rot fungi which were collected from various woody substrates in Korea were obtained from Korea University Culture (KUC) collection, Korea. The isolates were identified based on their morphological and molecular characteristics. Macro morphologies of the isolates on malt extract agar (MEA) and potato dextrose agar (PDA) media were evaluated and then the isolates were then grouped based on the results of morphological analysis. Subsequently, sequences of rDNA large subunit (LSU), internal transcribed spacer (ITS), or RNA polymerase II (RPB2) regions were analyzed to identify the isolates. In order to separate the isolates into brown-rot fungi and white-rot fungi, remazol brilliant blue R (RBBR) test and Bavendamm test were conducted. Based on the molecular information and results of RBBR and Bavendamm tests, a total of 87 isolates including 17 genera and 26 species and 222 isolates including 46 genera and 73 species were confirmed as brown-rot fungi and white-rot fungi, respectively. Therefore, brown rot fungi including genera Antrodia, Coniohora, Crustoderma, Cryptophorus, Daedalea, Fomitopsis, Gloeophyllum, Oligo porus, Platyloea, Polyporales, Pycnoporus, Tapimella, Tilletiopsis, and Tulasnella and white rot fungi including genera Amphinema, Antrodiella, Bjerkandera, Ceriporia, Cerrena, Coprnellus, Crepidotus, Cylindrobasidium, Daedaieopsis, Dendrocorticium, Dentipellis, Fomes, Fuscoporis, Heterobasidion, Hyphodintia, Hypholoma, Hypochnicium, Irpex, Lenzites, Megalocystidium, Meruliopsis, Microporus, Mucronella, Mycoaciella, Pachykytospora, Panellus, Peniophora, Peniophorella, Perenniporia, Phanerochaete, Phlebia, Phlebiella, Phlebiopsis, Phyllotopsis, Pleurotus, Polyporus, Porostereum, Pseudochaete, Radulomyces, Rhizochaete, Schizophyllu, Schizopora, Sistotrema, Skeletocutis, Stereum, Trametes, Trichaptum, and Tyromyces were used throughout this research.
Basic growth and physiological properties of the above isolates were evaluated prior to application of the isolates for mycoremediation. Optimal growth temperatures of each isolates were individually investigated and the evaluation of growth rates of the isolates under the optimal growth temperatures was also performed. Most of the isolates showed the highest growth rate under 30℃, followed by 25℃ and 35℃. Of the isolates, genus Phanerochaete showed the highest growth rate in the present study.
Production of organic acid by 87 isolates of brown-rot fungi was initially detected by pH measurement of fermentation broth after 7-day shaking incubation of the isolates in the malt extract liquid media. One isolate of A. alb ida, 1 isolate of A. sinuosa, 4 isolates of D. dikinsii, 9 isolates of F. palustris, 1 isolate of F. pinicola, and 2 isolates of Polyporales sp. increased in acidity of the fermentation broth under pH 3 by production of organic acid, the results of analysis of organic acid by high performance liquid chromatography (HPLC) revealed that all organic acid produced by the isolates was oxalic acid. A brown-rot fungus A. albida KUC8802 produced the highest amount of oxalic acid of up to 1,954 mg/L among all isolates.
Enzyme assay was also conducted to determine activities of cellulolytic enzymes [i.e., Filter paper assay (FPA)] and ligninolytic enzymes [i.e., laccase (Lac), manganese dependent peroxidase (MnP), and lignin peroxidase (LiP)]. In case of FPA, the highest activity (0.204 FPU/mL) was obtained by D. dickinsii KUC9181; FPA secreted by D. dickinsii KUC9181 showed higher activity than that by Trichorderma reesei which is well-known fungus to produce high amount of cellulolytic enzymes. A total of 14 isolates including genera Cerrena, Fornes, Hypholoma, Hypochnicium, Mycoaciella, Phlebia, and Trametes, 14 isolates including genera Dendrocorticium, Hypholoma, Irpex, Phanerochaete, and Phlebia, and 22 isolates including genera Amphinema, Bjerkandera, Ceriporia, Dentipellis, Heterobasidion, Hypochnicium, Irpex, Megalocystidium, Phlebia, Porostereum, Schizopora, and Stereum showed significant activities of Lac, MnP, and LiP, respectively. Differentially expressed genes by Cu in a brown-rot fungus Polyporales sp. KUC9061 which showed notable resistance to Cu were investigated using GeneFishing technology to better understand the molecular and physiological mechanisms of resistance of the isolate to Cu. The presence of Cu in MEA media decreased the fungal growth rate in a concentration dependent manner, but the fungal biomass was significantly increased.Increased expression of the genes encoding for the GIS2 DNA-binding protein and the 40S ribosomal protein S3A appears to be involved in this process. Oxalic acid is not used as a defense mechanism against high copper exposure, and ATP citrate lyase is not directly involved in oxalic acid production in this fungus. Several Cu-sensitive proteins showed stable gene expression, suggesting that mechanisms that do not rely on these genes are responsible for the Cu tolerance of the fungus.
The investigation of mycoremediation characteristics of the wood rot fungi can be categorized into the screening of useful fungi for mycoremeidiation and the application of the selected fungi to removal of pollutants. As the first research, of the 309 isolates of the wood rot fungi, candidates for mycoremediation of various pollutants were selected. And then, feasibility of mycoremediation by the selected fungi to apply for actual pollutants in the environment [i.e., wastewater polycyclic aromatic hydrocarbons industrial dyes] was evaluated.
containing heavy-metals, metal contaminated soils, (PARs) contaminated soil, wastewater containing For the biosorption of heavy-metals (i.e., Cu, Pb, and Cd), living wood rot fungi which was resistant to each heavy-metal and dead cell of wood rot fungi which have a high sorption capability to the heavy-metal were initially screened as biosorbent. The living state of A. sinuosa KUC8202 removed of up to 99.1% Cu and 90.3% Ph from solution containing each heavy-metal. It was confirmed that Cd ion in the solution was completely removed by living S. commune KUC8703. On the other hand, in the biosorption process of heavy-metals using dead cell of wood rot fungi, C lacerata KUC8111 was selected as biosorbent for Cu and Cd, and T. chioneus KUC8882 for Pb. It is anticipated that handling of dead cell was easier than that of living cell in terms of production of fungal biomass and contamination by other microorganism. Thus, dead cells from 2 isolates, C lacerata KUC8111 and T. chioneus KUC8882 were finally selected as biosorbent to apply for biosorption of heavy-metals from industrial wastewater. Metal removal rates from the wastewater were slightly decreased when compared with those from artificial wastewater, suggesting that biosorption capacities were hindered by other metals or organic matters in real wastewater.
A brown-rot fungus A. albida KUC8802 which was confirmed to produce the highest amount of oxalic acid among the all isolates in this research was used for bioleaching of metals from arsenic contaminated soils in 2 abandoned mine sites, Bok Su (BS) and Dae Yang (DY) mines, located at Jecheon, Korea. A second-order central composite design (CCD) with response surface methodology (RSM) was used to optimize bioleaching condition. Models evaluated showed that insignificant values for lack of fit (BS soil, p = 0.058; DY soil, p = 0.099) and high coefficients of determination (BS soil, r² = 0.97; DY soil, r² = 0.97) indicated that the models can predict the As removal rate with high accuracy under various extracting conditions. Under the optimal condition for As removal, oxalic acid produced by A. albida KUC8802 removed above 95% As from both BS and DY soils. In addition, other metal elements, Cd (64.7-86.6%), Cr (28.8-34.2%), Cu (44.6-56.1%), Ni (11.9-36.8%), Pb (4.0-35.8%), and Zn (20.8-42.4%), were concomitantly removed during the bioleaching process.
Four types of PARs [i.e., phenanthrene (PRE), anthracene (ANT), fluoranthene (FLT), and pyrene (PYR)] were used for screening of PARs resistant fungi, and 7 isolates showed more than 90% of resistance to PARs: M. bispora KUC8201, P. cinerea KUC9140, Peniophora sp. KUC8836, Phonerochaete sp. KUC8073, P. tremellosa KUC9161, Rhizochaete sp. KUC8357, and T. abietinum KUC9130. Except for Rhizochaete sp. KUC8357, the isolates showed higher degradabilities of PARs than P. chrysosporium KCTC6293. A white-rot fungus Peniophora sp. KUC8836 which showed high MnP activity during the biodegradation process degraded up to 86.5%, 77.4%, and 82.6% of PRE, FL T, and PYR, respectively. It is confirmed that Peniophora sp. KUC8836 decreased a total concentration of P AHs in sterilized soils near in creosote-treated crosstie 229.49 mg/kg to 32.93 mg/kg.
Of the 222 white-rot fungi, 25 isolates including 4 isolates of B. adusta, 5 isolates of C. lacerata, 3 isolates of 1. lacteus, 1 isolate of P. jraxinea, 10 isolates of Phanerochaete spp., 1 isolate of P. radiata, and 1 isolate of P. spadiceum were initially selected by investigation of ability to decolorize chemically different synthetic dyes (i.e., Conge red, Reactive Blue 4, and Orange II) in solid medium. Four isolates of white-rot fungi, B. adusta KUC9065, C. lacerata KUC8090, P. calotricha KUC8003, and P. spadiceum KUC8602 were finally selected on the strength of their ability to decolorize synthetic dyes in liquid medium, and were used to decolorize industrial effluents. A white-rot fungi B. adusta KUC9065 showed the highest decolorizing rates, resulting in increase in the transmittance of visible light by 71 - 92%. Biodecolorization of wastewater by B. adusta KUC9065 could be attributed to the MnP and Lac produced by the fungus. Furthermore, it was confirmed that the acute toxicity of wastewater to Daphinia magna decreased from 2.5 to 2.1 and from 3.5 to 2.6 toxic units over 24 and 48 hours, respectively.
Mycoremediation is the employment of fungal species for bioremediation. Despite of recent advances in mycoremediation, a knowledge database system for mycoremediation is not available yet. In order to provide a taxonomic resource and molecular function-oriented portal for mycoremediation, we developed a wiki-based mycoremediation information system (http://mycopedia.org)includingfungalspecieslistedintheKUC.This system will serve as a valuable resource to those who aim to study and use fungi for a bioremediation tool. We also developed an algorithm for a functional categorization of fungal strains and categorized 309 KUC fungal species by experimental results (e.g., optimal growth conditions, pH, Enzyme activities, metal resistance, absorption of metal, PAH resistance and ability of degradation of dyes). For a potent fungal species (C. lacetera), we attempted whole genome sequencing using NGS and RNAseq and all data will be integrated the wiki-base information portal.

목차 Contents

• 표지 ... 1
• 제출문 ... 2
• 보고서 요약서 ... 3
• 요약문 ... 4
• SUMMARY ... 11
• CONTENTS ... 15
• 목차 ... 16
• 제1장 연구개발과제의 개요 ... 17
• 1. 연구개발의 필요성 ... 17
• 2. 연구개발의 목표 및 내용 ... 20
• 제2장 국내외 기술개발 현황 ... 23
• 1. 연구개발 대상기술의 국내 수준 ... 23
• 2. 연구개발 대상기술의 국외 수준 ... 23
• 3. 향후 전망 ... 24
• 제3장 연구개발수행 내용 및 결과 ... 25
• 제1절 국내 자생 목재미생물의 기초 특성 규명 ... 25
• 제2절 국내 자생 목재미생물의 mycoremediation 특성 규명 ... 63
• 제3절 Mycoremediation 용 환경생물자원 종합정보시스템구축 ... 136
• 제4장 목표달성도 및 관련분야에의 기여도 ... 170
• 1. 연구개발 목표의 달성도 ... 170
• 2. 관련분야의 기술발전에의 기여도 ... 171
• 3. 연구 성과 ... 172
• 제5장 연구개발결과의 활용계획 ... 177
• 1. 연구개발결과의 활용방안 ... 177
• 2. 기대성과 ... 178
• 3. 추가연구의 필요성 ... 179
• 제6장 연구개발과정에서 수집한 해외과학기술정보 ... 180
• 1. Mycoremediation 용 주요 목재미생물 ... 180
• 2. 주요 국가의 균류 수집기관 및 특화데이터베이스 ... 182
• 제7장 참고문헌 ... 183
• 제8장 첨부자료 ... 189
• 1. 공시균주의 DNA sequences 정보 ... 189
• 2. 공시균주의 배지별 생장형태 ... 252
• 끝페이지 ... 275