미생물을 이용한 축사 파리 및 암모니아 저감 기술의 개발

발 표 자

건국대학교 송 민 동

2003 년도 충북지역환경기술개발센터 최종 평가회 발표자료

2004. 01. 13

Introduction

축사의 파리는 가축 질병을 전염시키는 주요 매개체일뿐만 아니라

가축 및 사람에게 스트레스를 주는 주요 원인

가축 호흡기 발생의 주요 원인인 가축분뇨로부터의 암모니아 저감

필요함 .

지역특성상 환경친화적 축산 요구됨 .

연구배경

축산분뇨에서 발생하는 암모니아는 악취의 가장 큰 원인

가축의 호흡기 질병의 원인인 악취제거 필요

파리 발생 억제에 따른 질병 사전예방 필요

항생제 사용 감소로 환경친화적 축산환경조성 필요

충북지역 특성상 환경친화적 축산이 절대적으로 필요

연구의 필요성

연구목적

축사 파리발생 억제를 위한 미생물의 분리 및 특성 조사

축사 암모니아 발생 저감을 위한 미생물의 분리

미생물을 이용한 축사 파리 및 암모니아 저감 기술의 개발

일본의 미생물학자가 병든 누에 (Bombyx mori) 로부터 Bacillus 를 분리 (Bacillus thuringiensis : Bt)

Gram-positive bacteria

결정형태의 insecticidal crystal protein 생산

δ-endotoxin ; ICPs(Insecticidal crystal proteins)

* Cry1, Cry2, Cry3, Cry4 - Toxic

* cytolysin(cyt) – Non-Toxic

Bacillus thuringiensis 의 δ-endotoxin 에

관한 연구 현황

균주의 특성

Bacillus thuringiensis 의 δ-endotoxin 에 관한 연구 현황

연구기작 및 동향

살충 특이성을 가진 결정체 단백질의 생산은 포자형성 시

서로 다른 단계동안 RNA polymerase 의 시그마 인자와 함께 , 영양성장과 포자형성 자극

포자형성의 개시는 세포내의 GTP 농도에 의해 조절

δ-endotoxin 의 생성은 유도성을 지님

현재 국내외적으로 내독소 단백질 분해 미생물에 관한 연구가 활발히 진행 중

지금까지 주된 연구 방향은 주로 나비목에 치중되어져 있다

파리목에 관한 연구 및 미생물 제제화 연구는 아직 개발단계

토양으로부터 유용 균주의 분리

분리 균주의 특성조사

성장곡선

분리 균주의 동정

생화학적 특성

전자현미경을 이용한 형태학적 특성

다양한 항생제에 대한 최소억제농도 (MIC) 조사

delta-toxin 의 정제 및 특성

정제산물

연구내용1

cry gene profile 분석

Endospore 및 crystal protein 생산조건 확립

분리 균주의 효소활성 측정

Multiplex PCR

Crystal protein 특성조사

SDS-PAGE

2

0.1M sodium acetate in 10ml NB

Soil 1g

vortex Shaking incubation for 6hr at 30℃

Heating for 10min at 100℃

Spreading on GYS medium

Incubation for 24hr at 30℃

Bacillus thuringiensis strain selection

Spreading on nutrient agar (NA)

Subcultured Bacillus thuringiensis k-1

Isolation of Bacillus thuringiensis Strain

Insect Bioassay for B. thuringiensis Selection

M. Domestica (larvae) were obtained from a pig farm near Chungju city (Korea)

Subcultured Bacillus thuringiensis k-1 Inoculation in 10ml

NB mediumShaking incubation at 37℃ Shaking incubation

for 3days at 30℃ in GYS medium (until 107 to 108 spores/ml)

1ml

Centrifugation at 10,000g for 20min at 4℃

pellets

supernatant

Three times washing with dH2O and sample dilution (10 배 , 100 배 )

Insect Bioassay against fly larvae (M. domestica)

B. thuringiensisB. thuringiensis str strains ains

No. of larvae No. of larvae tested tested

No. of the dead No. of the dead Mortality (%) Mortality (%)

Control Control 3030 00 00

Bt k-1 Bt k-1

(original amount) (original amount) 3030 2020 66.766.7

Bt k-1 Bt k-1 (supernatant ) (supernatant )

3030 2525 83.3 83.3

Larvae mortality was determined after incubation at room temperature (25±2 ) for 72h ℃Larvae mortality was determined after incubation at room temperature (25±2 ) for 72h ℃using 10using 1055 to 10 to 1066 spores/ml.  spores/ml. 

Table 1. Bio-toxicity of Bacillus thuringiensis k-1 against fly larvae (M. domestica)

Growth curve of Bacillus thuringiensis k-1

Use of Media : Nutrient Broth (NB, 0.3% beef extract, 0.5% peptone)

Culture Condition : 48hr, 37℃

Bacterial Growth Curve

1.0E+00

1.0E+01

1.0E+02

1.0E+03

1.0E+04

1.0E+05

1.0E+06

1.0E+07

1.0E+08

1.0E+09

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36

시간

(CFU

/ml)

생균

수

Fig. 1. Growth curve of Bacillus thuringiensis strain k-1

Stationary phase

Biochemical Characteristics of B. thuringiensis k-1

Use of Identification Kit : API CHB 50 kit and Medium

Culture Condition : 24hr, 37℃

+Gelatin hydrolase

+Nitrate reduction

-Indole production

-Tryptophan deaminase

-Urease

-H2S production

+Citrate utilization

-Ornithine decarboxylase

-Lysine decarbosylase

-Arginine dihydrolase

+Beta-Galactosidase

+Acid from glucose

+Voges-Proskauer

+Catalase

+Spore formation

RodCell form

+Motility

+Gram staining

Bacillus thuringiensis k-1 Characteristics

Table 2. Morphological and biochemical properties of Bacillus thuringienis k-1

Table 3. Biochemical characteristics (carbohydrates) of Bacillus thuringiensis k-1 page 1

Characteristics B. thuringiensis k-1 Cahracteristics B. thuringiensis k-1

Glycerol + Salicine +

Erythritol - Cellobiose +

D-Arabinose - Maltose +

L-Arabinose - Lactose -

Ribiose + Melibiose -

D-Xylose - Saccharose +

L-Xylose - Trehalose +

Adonitol - Inuline -

Beta-Methyl-xyloside - Melezitose -

Galactose - D-Raffinose -

D-Glucose + Amidon +

D-Fructose + Glycogen +

D-Mannose + Xylitol -

L-Sorbose - Beta-Gentiobiose -

page 2

Characteristics B. thuringiensis k-1 Cahracteristics B. thuringiensis k-1

Rhamnose - D-Turanose -

Dulcitol - D-Lyxose -

Inositol - D-Tagatose -

Mannitol - D-Fucose -

Sorbitol - L-Fucose -

Alpha-Methyl-D-mannoside

- D-Arabitol -

Alpha-Methyl-D-Glucoside

- L-Arabitol -

N Acetyl glucosamine + Gluconate -

Amygdaline + 2 aceto-gluconate -

Arbutine + 5 aceto-gluconate -

Esculine +

+, Positive; -, Negative

Electron Microscopy (TEM, Transmission Electron Micrographs)

Strain k-1 was grown for 3days in GYS medium at 30 ℃

Sample was blocked in 4% agar

Sample was fixed with Karnowsky`s fixative and then postfixed in 1% OsO4

Sample was dehydrated through an ethanol-propylene oxide series and embedded in Epon 812

Thin sections were cut on ultramicrotome, stained with 1% uranyl acetate and lead citrated

Photographed

Fig. 2. Transmission electron micrographs of sporulating of Bacillus thuringiensis k-1 amplified 25,000× (A). Parasporal crystal (c) and spore (e).

The test was performed with the use of serial 2-fold dilutions of each antibiotic as described by Cleeland et al.

The resistance of Bacillus thuringiensis k-1 against antibiotics was examined after cultivati

on for 18hr at 37 .℃

Inoculation of Bacillus thuringiensis

1.95ug 0ug

1.95ug 0ug

1000ug/ml500ug/ml250ug/ml125mg/ml62.5ug/ml 31.3ug 15.6ug 7.8ug 3.9ug

Shaking incubate, 18hrs, 37℃

1000ug/ml500ug/ml250ug/ml125mg/ml62.5ug/ml 31.3ug 15.6ug 7.8ug 3.9ug

MIC value

antibiotics

Antibiotics susceptibility of B. thuringiensis k-1

Strain k-1Strain k-1 Kurstaki Kurstaki (HD-1)(HD-1) Israelensis Israelensis (HD-522)(HD-522)

AmpicillinAmpicillin 62.562.5 100100 100100

KanamycinKanamycin <1.95<1.95 12.512.5 3.93.9

OxacillinOxacillin 62.562.5 250250 7.87.8

ColistinColistin 31.331.3 125125 62.562.5

TetracyclineTetracycline 7.97.9 6.256.25 3.93.9

Penicillin GPenicillin G 125125 >1000>1000 >1000>1000

ErythromycinErythromycin 250250 <1.95<1.95 <1.95<1.95

NeomycinNeomycin 7.97.9 3.93.9 3.93.9

Table 4. Determination of minimum inhibitory concentration of various antibiotics against B. thuringiensis k-1, B. thuringiensis subsp. kurstaki (HD-1) and B. thueingiensis subsp. israelensis (HD-522).

Purification of insecticidal proteins from strain k-1

B. thuringiensis was grown for 3 days in the GYS medium at 30 and 180rpm until complete autolysis was achieved.℃

Purification crystal proteins was carried out by discontinuous sodium bromide (NaBr) gradients of 30% to 70%.

Fig. 3. SDS-PAGE of Bacillus thuringiensis k-1 crystal proteins purified. M, molecular marker; arrows indicate Cry proteins of ~130, ~80, ~60kDa.

130kD

80kD60kD

Multiplex PCR for rapid determination of cry genes

The extended multiplex PCR screening is a rapid method for detection and differentiation of Bacillus thuringiensis field strains and for prediction their insecticidal activities in order to direct them for subsequent toxicity assays against Lepidoptera, Coleoptera, and Diptera.

The 10 primers contained eight forward primers and two reverse primers for cry1 gene determination used in this study.

To identify cry4, one forward and one reverse primer used in reaction.

Composition Vol. unit Temp. Time cycle

Template 15ul 95 2min 1cycle

Primer (F) each 1ul 8ul 200uM

Primer (R) each 1ul 2ul 200uM 95 1min

10X buffer 8ul 52 2min 30cycle

Ex Taq. 0.5ul 0.5U 72 3min

2.5mM dNTPs 4ul 200uM

dH2O 12.5ul 72 7min 1cycle

Total volume 50ul

Table 5. The optimal conditions of polymerase chain reaction (PCR).

cry1A(c), 487bpcry1D, 414bp

cry1A(b), 238bp

1 2M M

cry1A(c), 487bpcry1D, 414bp

cry1A(b), 238bp

1 2M M

Fig 4. PCR survey of B. thuringiensis k-1 and B. thuringiensis subsp. kurstaki for cry1 contents. Total DNA samples from B. thuringiensis k-1 and B. thuringiensis subsp. kurstaki were analyzed by PCR with a mixture of cry1 specific primers. Lane M: Bio basic 100bp-1kb ladder; lane 1, B. thuringiensis k-1; lane 3, B. thuringiensis subsp. kurstaki ;  M: Bio basic 100bp-1kb ladder

Culture conditions for viable cell growth and endospore formation (crystal protein)

in 10ml various media

Shaking incubation for 8h, 12h and 16h at 30℃

2. Heating for 10min at 100℃

Spreading on NA medium

Incubation for 12h at 30℃

Inoculation of Bacillus thuringiensis k-1

1. Direct spreading

Total and endospore forming cell counting

Time Media Viable cell Endospore

NB 2.0x108 5.8x104

MED2 1.2x108

8 BHI 4.0x108

GYS 4.6x104

PGY 2.4x108

NB 1.4x108 4.0x105

MED2 1.8x108

12 BHI 2.6x108 1.8x105

GYS 8.2x106 5.3x105

PGY 1.3x108

NB 3.6x106 1.7x106

MED2 6.4x105

16 BHI 1.1x107

GYS 1.7x107 2.0x106

PGY 1.5x106

Table 6. Viable cell and endospore forming cell in various culture conditions ( CFU/ml )

Enzyme activities of Bacillus thuringiensis k-1

Enzyme Substrate (basal medium: NA1) Dye Identification

Protease 2%(w/v) skim milk - Clear zone

Cellulase(CMCase) 1%(w/v) CMC2 0.2%(w/v) congo red sol. Clear zone

Amylase 1%(w/v) soluble starch 0.2%(w/v) I2+2%(w/v) KI sol. Clear zone

Xylanase 1%(w/v) oat spelt xylan - Clear zone

Table. 7 Screening media and dyes used for the detection of various enzyme activities

11Nutrient agar (0.3% beef extract, 0.5% peptone, 1.5% agar)Nutrient agar (0.3% beef extract, 0.5% peptone, 1.5% agar)22Carboxymethyl cellulose (medium viscosity)Carboxymethyl cellulose (medium viscosity)

Table. 8 Enzyme activities of Bacillus thuringiensis k-1

Items Xylasnase Protease Cellulase Amylase

Enzyme activity ++++ ++ + ++

Size of clear zone(mm)

(Width of colony / Width of clear zone)

6.5/12.0 5.0/9.0 5.0/5.5 5.0/10.0

++++ represents excellent clear zone formation; ++ good; + fair++++ represents excellent clear zone formation; ++ good; + fair

암모니아 저감을 위한 복합효소 생산

미생물의 분리 및 동정

토양으로부터 유용 균주의 분리 및 선발

분리 선발 균주의 특성 조사

분리 선발 균주의 동정

형태학적 특성

효소활성 측정

생화학적 특성

Isolation of the strain having multi-enzyme activities

in 9ml 0.85% NaCl

Soil 1g

vortexing Shaking incubation for 6hr at 37℃

Spreading on nutrient agar medium

Incubation for 24hr at 37℃

Identification

Inoculation on enzyme screening mediums (amylase, cellulase, and protease)

Clear zone detection Incubation for 12hr at 37℃

Enzyme activities of Bacillus sp. strains

Enzyme Substrate (basal medium: NA1) Dye Identification

Protease 2%(w/v) skim milk - Clear zone

Cellulase(CMCase) 1%(w/v) CMC2 0.2%(w/v) congo red sol. Clear zone

Amylase 1%(w/v) soluble starch 0.2%(w/v) I2+2%(w/v) KI sol. Clear zone

Table. 9 Screening media and dyes used for the detection of various enzyme activitiesTable. 9 Screening media and dyes used for the detection of various enzyme activities

1Nutrient agar (0.3% beef extract, 0.5% peptone, 1.5% agar)2Carboxymethyl cellulose (medium viscosity)

Fig 5. Protease, cellulase, and amylase activities of 1Fig 5. Protease, cellulase, and amylase activities of 1 stst isolated strains isolated strains

Enzyme activity3

Strain Growth1,2 at 37℃ CMCase Protease Amylase

2-4 +++ + - ++

2-5 +++ - ++ ++

3-1 +++ - ++ +

3-3 +++ - ++ +++

5-1 +++ +++ +++ +++

6-7 +++ +++ +++ ++

6-9 +++ ++ +++ ++

7-3 +++ ++ ++ ++

7-10 +++ - ++ +++

7-12 +++ - ++ +++

8-5 +++ ++ ++ ++

8-7 ++ + - ++

9-1 + - - +

9-3 + - - -

9-6 ++ - ++ -

10-4 + - - -

Table 10. Enzyme activities of 1st-selected microorganisms

11The rate of growth was evaluated after 12hr on nutrient agarThe rate of growth was evaluated after 12hr on nutrient agar22+++ represents excellent growth+++ represents excellent growth33+++ represents excellent clear zone formation on screening medium+++ represents excellent clear zone formation on screening medium ++; good, +; fair, -; no clear zone formation ++; good, +; fair, -; no clear zone formation

Fig 5. Protease, amylase, and cellulase activities of the finally isolated 5-1 Fig 5. Protease, amylase, and cellulase activities of the finally isolated 5-1

Protease Cellulase

Amylase

Identification of the selected strain

Use of Identification Kit : API CHB 50 kit and Medium

Result: Bacillus subtilis

+Gelatin hydrolase

+Nitrate reduction

-Indole production

-Tryptophan deaminase

-Urease

-H2S production

+Citrate utilization

-Ornithine decarboxylase

-Lysine decarbosylase

-Arginine dihydrolase

+Beta-Galactosidase

+Acid from glucose

+Voges-Proskauer

+Catalase

+Spore formation

RodCell form

+Motility

+Gram staining

5-1 Characteristics

Table 11. Morphological and biochemical properties of 5-1

Table 12. Biochemical characteristics (carbohydrates) of 5-1 page 1page 1

Characteristics 5-1 Cahracteristics 5-1

Glycerol + Salicine +

Erythritol - Cellobiose +

D-Arabinose - Maltose +

L-Arabinose + Lactose -

Ribiose + Melibiose +

D-Xylose + Saccharose +

L-Xylose - Trehalose +

Adonitol - Inuline +

Beta-Methyl-xyloside - Melezitose -

Galactose - D-Raffinose +

D-Glucose + Amidon +

D-Fructose + Glycogen +

D-Mannose + Xylitol -

L-Sorbose - Beta-Gentiobiose -

page 2

Characteristics 5-1 Cahracteristics 5-1

Rhamnose - D-Turanose +

Dulcitol - D-Lyxose -

Inositol + D-Tagatose -

Mannitol + D-Fucose -

Sorbitol + L-Fucose -

Alpha-Methyl-D-mannoside

- D-Arabitol -

Alpha-Methyl-D-Glucoside

+ L-Arabitol -

N Acetyl glucosamine - Gluconate -

Amygdaline + 2 aceto-gluconate -

Arbutine + 5 aceto-gluconate -

Esculine +

+, Positive; -, Negative

파리유충에 활성을 나타내는 δ-endotoxin 생산 B. thuringiensis 균주 분리 및 동정 ( 형태학적 , 생화학적 특성조사 ) 완료

δ-endotoxin 단백질 정제 , 특성조사 및 보유 유전자 검색 완료

파리유충 외피분해를 위한 chitinase 생산 미생물 분리 확보

결 론 [PART I]

미생물을 이용한 축사 파리 저감 기술의 개발

축 분뇨의 암모니아 발생 저감을 위해 우선적으로 각종 효소활성이 높아 사료첨가 시 사료의 단백질 이용효율 제고를 통한 축 분내 암모니아 발생 저감을 목적으로 하는 미생물 균주 분리

효소활성이 가장 우수하며 urease 활성이 없는 (암모니아로의 전환 없음 ) 5-1 균주 최종선발 및 동정완료 (Bacillus subtilis)

암모니아의 질산화에 관여하는 Denitrobacter sp. 및 암모니아와 함께 분뇨냄새의 주 원인물질인 휘발성지방산과 이산화황을 이용하는 광합성세균 (photosynthetic bacteria) 확보

미생물을 이용한 암모니아 저감 기술의 개발

결 론 [PART II]

본 과제 성과물

논문 1 편 ( 한국 학술진흥재단 등재학술지 이상 )

특허출원 ( 진행 중 )