Production and characterization of exopolysaccharides produced by an entomopathogenic fungus, Paecilomyces tenuipes C240 : 눈꽃 동충하초(Paecilomyces tenuipes C240)의 액체배양에 의한 세포외 다당체 생산과 특성

국문 초록 (Abstract)

본 연구에서는 눈꽃동충하초 (Paecilomyces tenuipes C240)의 액채배양을 통하여 세포외 다당체를 생산하고 그 특성을 고찰하였다. 눈꽃동충하초의 균사체와 세포외 다당체의 생산 최적화는 one-factor-at-a-time 및 orthogonal matrix method를 이용하였다. 첫번째로 one-factor-at-a-time method는 균사체와 세포외 다당체 생산을 위하여 배지조성 (탄소원, 질소원, 미량원소)과 외부 환경적 요소 (초기 pH, 온도)의 영향을 최적화하기 위해 적용하였다. 그 결과 최대 균사체와 세포외 다당체 생산을 위한 최적 탄소원, 질소원 그리고 미량원소는 각각 glucose, KNO₃, K₂HPO₄와 MgS O₄이었으며, 최적 온도와 초기 pH는 각각 28℃와 6.0으로 나타났다. Glucose, , KNO₃, K₂HPO₄와 MgS O₄의 최적 농도는 orthogonal matrix method를 이용하여 구하였다. 눈꽃 동충하초의 균사체에 대한 배지 조성의 영향은 Glucose〉K₂HPO₄〉KNO₃〉MgS O₄의 순서로 나타났으며 세포외 다당체 생산에 있어서는 glucose〉 K₂HPO₄〉MgS O₄〉KNO₃의 순서였다. 그리고 눈꽃 동충하초의 균사체 생산을 위한 최적농도는 glucose (4%), KNO₃(0.6%), K₂HPO₄(0.1%)와 MgS O₄·5H₂O (0.1%)이었고 세포외 다당체 생산을 위한 최적농도는 glucose (3%), KNO₃(0.4%), K₂HPO₄(0.1%)와 MgS O₄·5H₂O (0.1%)이었다. 이 모델의 타당성 연구를 수행향 결과, 최적화 이전의 발효결과에 비해 균사체 및 다당체생산량이 크게 증가하였는데, shake flask culture에서는 균사체 10.18 g/l와 세포외 다당체 1.89 g/l를 얻었으며, 5-L 교반 발효조에서는 세포외 다당체 2.36 g/l를 생산하였다.
세포외 다당체 생산에 있어서 교반 속도의 영향을 교반조형 발효조 (stirred-tank reactor, STR)와 공기부상 발효조 (airlift reactor, AR)에서 각각 수행하였는데, 최적 교반 속도는 150 rpm이었으며 이때 다당체 최종농도 2.33 g/l와 비생산속도 (specific production rate)는 0.312 g/g/h이었다. 그러나 최대 균사체 생산 (21.06 g/l)은 높은 교반 속도인 300 rpm에서 관찰되었다. 세포외 다당체의 비생산속도는 공기부상 발효조에서 0.456 g/g/h로써 교반조형 발효조보다도 높은 값을 나타내었다. 균체가 제거된 발효상등액을 Sepharose CL-6B를 이용하여 분획한 결과, STR에서는 세 종류의 세포외 다당체 (STR-I, II, III)를, AR에서는 두 종류의 세포외 다당체 (AR-I, II)를 얻을 수 있었다. STR-I, STR-II, STR-III, AR-I와 ARII의 분자량은 각각 1820, 25, 1.8, 1160과 6.7 kDa로 관찰되었다.
균사체 성장과 세포외 다당체 생산에 미치는 통기영향 실험에서는 높은 통기 속도인 3.5vvm에서 최대 균사체 생산량 (21.56 g/l)과 세포외 다당체량 (2.36 g/l)을 얻을 수 있었다. 세포외 다당체를 Sepharose CL-6B를 이용하여 분획한 결과 세 종류의 다당체 (Fr-I, Fr-II, Fr-III)가 나타났으며 각각 1,820, 14-45.4, 1.8-1.9 kDa의 분자량을 가지는 것으로 나타났다. 생산된 세포외 다당체의 성분분석 결과, 탄수화물 76.6-84.4와 단백질 15.6-23.4%의 조성을 갖는 단백다당체로 관찰되었다. 다양한 통기 속도 조건에서 생산된 다당체 중, Fr-III는 유사한 분자량과 화학적 조성을 가지는 데 비해 Fr-I과 Fr-II는 서로 다른 화학적 조성을 보였다.
눈꽃 동충하초의 세포외 다당체의 최대 생산을 위한 물리적 조건들 (pH, 교반속도, 통기속도)의 적합한 조합을 위하여 Uniform design을 이용하였다. 그 결과, 최대 균사체 생산 조건은 pH 4.88로 유지하고 통기 속도 2 vvm, 교반 속도 350 rpm 일 때 그리고 최대 세포외 다당체 생산 조건은 pH 4로 유지하고 통기 속도 2 vvm, 교반 속도 150 rpm 일 때가 가장 좋았다. 최적 조건에서 최대 3.39 g/l의 세포외 다당체를 5-L STR에서 얻을 수 있었다.

다국어 초록 (Multilingual Abstract)

Production and characterization of the exopolysaccharides (EPS) produced by submerged culture of an entomopathogenic fungus, Paecilomyces tenuipes C240 was studied. Process optimization of submerged culture conditions for the mycelial growth and EPS production by Paecilomyces tenuipes C240 was conducted by one-factor-at-a-time and orthogonal matrix methods. First, the one-factor-at-a-time method was adopted to investigate the effects of different variables of medium components (i.e., carbon, nitrogen, and mineral sources) and environmental factors (i.e., initial pH and temperature) for the mycelial growth and exopolysaccharides production. Among these variables, glucose, KNO₃, K₂HPO₄and MgS O₄ were identified to be the most suitable carbon, nitrogen, and mineral sources, respectively. The optimal temperature and initial pH for mycelial growth and EPS production were found to be 28℃ and 6.0, respectively. Subsequently, the concentration of glucose, KNO₃, K₂HPO₄and MgS O₄ were optimized using the orthogonal matrix method. The effects of media composition on the mycelial growth of P. tenuipes C240 were in the order of glucose〉K₂HPO₄〉KNO₃〉MgS O₄, and those on EPS production were in the order of glucose〉 K₂HPO₄〉MgS O₄〉KNO₃. The optimal concentration for the enhanced production are determined as 4% glucose, 0.6% KNO₃, 0.1% K₂HPO₄, and 0.1% MgS O₄·5H₂O for mycelial yield, and 3% glucose, 0.4% KNO₃, 0.1% K₂HPO₄, and 0.1% MgS O₄·5H₂O for EPS production, respectively. The subsequent verification experiments confirmed the validity of the models. This optimization strategy in shake flask culture leads to a mycelial yield of 10.18 g/l, and EPS production of 1.89 g/l, which were considerably higher than those obtained in the preliminary studies. Under optimal culture conditions, the maximum EPS concentration in a 5-1 stirred-tank bioreactor indicated 2.36 g/l.
The effect of shear force on the production of EPS was investigated in a stirred-tank reactor (STR) and an airlift reactor (AR). The optimal agitation rate for the production of EPS in the STR was 150 rpm with the mycelial morphology of hairy pellets, where the final concentration and the specific production rate of EPS were 2.33 g/l and 0.312 g/g/h, respectively. However, the maximum concentration of biomass (21.06 g/l) was obtained at a high agitation speed of 300 rpm in the STR. The specific production rate of EPS (0.456 g/g/h) in the AR was significantly higher than that achieved in the STR, in which the typical morphological form of mycelium was a loose clump. The three EPS groups in the STR(designated as STR-I, II, and III) and two groups of EPSs in the AR (designated as AR-I, and II) were obtained from the culture filtrates by a gel filtration chromatography on Sepharose CL-6B. The molecular weights of STR-I, STR-II, STR-III, AR-I, and AR-II were determined to be 1820, 25, 1.8, 1160, and 6.7 kDa, respectively. The carbohydrate composition in each EPS was quite different from each other: the major component was glucose (STR-I, III, and AR-I), mannose (STR-II), and arabinose (AR-II). On the other hand, no significant difference in amino acid composition was observed. It was demonstrated that the shear force affected the quality of microbial polysaccharides in terms of molecular weight and composition.
The effect of aeration on the production of EPS and their quality was studied. The maximum production of EPS (2.36 g/l) and mycelial biomass (21.56 g/l) were achieved at a high aeration rate of 3.5 vvm. There was a significant morphological difference in mycelial biomass at different aeration conditions: loose mycelial clumps developed and their size and hairiness increased as the aeration rate increased from 0.5 to 3.5 vvm, increasing the apparent viscosity of the broth. The three EPS groups (designated as Fr-I, Fr-II, and Fr-III) were fractionated from the culture filtrates by a gel filtration chromatography on Sepharose CL-6B and their molecular weights were estimated to be 1,820, 14-45.4, and 1.8-1.9 kDa, respectively. The compositional analysis revealed that all three EPS groups were proteoglycans consisting of 76.6-84.4% carbohydrates and 15.6-23.4% proteins. The group Fr-III had the same molecular weight and similar chemical composition, whereas the groups Fr-I and Fr-II showed quite different chemical compositions while produced by varying aeration conditions.
The uniform design, a statistical optimization technique, was employed for obtaining the best suitable combination of physical parameters, viz. pH (controlled), agitation intensity and aeration rate for maximum EPS production by P. tenuipes C240 in a batch bioreactor. The optimal combination for the enhanced production was determined as pH (controlled)-4.88, aeration-2 vvm and agitation-350 rpm for mycelial yield, and pH (controlled)-4, aeration-2 vvm and agitation-150 rpm for maximum EPS production. Under optimal culture conditions, the maximum EPS concentration obtained in a 5-1 stirred-tank bioreactor was 3.39 g/l.
The unstructured model of EPS production by fermentation with P. tenuipes C240 was developed in a batch system. The Logistic equation for mycelial growth, the Luedeking-Piret equation for EPS production and Luedeking-Piret-like equations for glucose consumptions were successfully incorporated into the model. Parameters for the model were determined based on experimental data using a program developed in Microsoft EXCEL. The value of the key kinetic constants were: maximum specific growth rate (㎛), 0.7281 1/h; growth associated constant for EPS production (α), 0.1743 g/(g cells); non-growth associated constant for EPS production (β), 0.0019 g(g cells); with maintenance coefficient (㎳), 0.0572 g/g. When compared with batch experimental data, the model appeared to provide a reasonable description for each parameter during the growth phase. The model showed that the production of EPS was growth-associated. In general, the proposed model appears to be useful for the design, scale-up, control and optimization of fermentation bioprocess.

목차 (Table of Contents)

• ABSTRACT = i
• CONTENTS = vi
• LIST OF TABLES = xi
• LIST OF FIGURES = xiii
• I. Introduction = 1
• 1. An overview = 1
• 2. Statistical optimization of submerged culture conditions for mycelial biomass and EPS productiion = 7
• 3. Effect of agitation intensity and aeration rate on the production of EPS in a stirred tank (STR) and airlift reactors (AR) = 8
• 4. Application of uniform design for optimization of the physical parameters for EPS production in a batch bioreactor = 9
• 5. Unstructure kinetic models of EPS production by batch fermentation of P. tenuipes C240 = 10
• II. The signification and objectives of this study = 12
• 1. Significance = 12
• 2. Objectives = 12
• III. Materials and Methods = 13
• 1. Chemicals = 13
• 2. Microorganism and media = 13
• 3. Inoculum preparation = 13
• 4. Seed culture = 14
• 5. Fermentations in bioreactor = 14
• 6. Analytical methods = 14
• 6.1. Mycelial dry weight and EPS quantification = 15
• 6.2. Estimation of residual sugar = 15
• 6.3. Measurements of rheology and morphology = 15
• 6.4. Purification of the EPS = 16
• 6.5 Analysis of carbohydrates and amino acids = 17
• IV. Results and Discussion = 22
• PART 1. Statistical optimization of submerged culture conditions for mycelial biomass and EPS production in shake flask culture = 22
• 1.1. One-factor-at-a-time method = 22
• 1.1.1. Effect of carbon sources = 22
• 1.1.2. Effect of nitrogen sources = 22
• 1.1.3. Effect of mineral sources = 23
• 1.1.4. Effect of initial pH and temperature = 23
• 1.2. Orthogonal matrix method = 24
• 1.2.1. Order of effects of factors = 24
• 1.2.2. Optimum levels of each factor = 25
• 1.3. Fermentation results = 26
• PART 2. Production of EPS by submerged culture in stirred-tank (STR) and airlift reactors(AR) = 36
• 2.1. Effect of agitation speed in STR = 36
• 2.2. Comparison of EPS and biomass production between STR and AR = 38
• 2.3. Broth rheology in the STR = 40
• 2.4. Comparison of molecular and chemical properties of EPS = 41
• PART 3. Influence of aeration on the production and the quality of the EPS in a stirred-tank fermenter = 53
• 3.1. Effect of aeration rate on the fermentation kinetics = 53
• 3.2. Effect of the aeration rate on the fungal morphology and broth rheology = 56
• 3.3. Effect of aeration on the molecular properties of EPS = 58
• 3.4. Conclusions = 59
• PART 4. Application of uniform design for optimization of the physical parameters for EPS production by P. tenuipes C240 in a batch bioreactor = 69
• 4.1. Optimization of the physical parameters for EPS production in a batch bioreactor = 69
• 4.2. Fermentation results = 72
• PART 5. Unstructure kinetic models of EPS production by batch fermentation = 77
• 5.1. Kinetic model = 77
• 5.1.1. Microbial growth = 77
• 5.1.2. Product formation = 78
• 5.1.3. Glucose consumption = 79
• 5.2. Kinetic model for EPS = 80
• 5.2.1. Microbial growth = 80
• 5.2.2. Product formation = 81
• 5.2.3. Substrate Uptake = 81
• 5.2.4. Testing the model = 82
• 5.3. Conclusions = 82
• V. References = 91
• ABSTRACTS(In Korean) = 107
• Curriculum Vitae = 110