Characterization of a Flocculent Yeast, Candida tropicalis HY200 and Xylitol Production Using Repeated-Batch Fermentation
- 발행기관 The Graduate School of Ajou University
- 지도교수 Ryu, Yeon-Woo
- 발행년도 2005
- 학위수여년월 2005. 2
- 학위명 박사
- 학과 및 전공 일반대학원 공학계열
- 본문언어 영어
초록/요약
On the basis of high osmotic tolerance and xylitol production, a novel flocculent yeast strain was screened from soils of rice paddy. The strain has a highly conserved relationship to Candida tropicalis and was designated Candida tropicalis HY200. HY200 steadily flocculated during and/or after growing in the formulated medium. It was found to separate in culture broth and to flocculate easily at the stationary phase. Here flocculation of Candida tropicalis HY200 was systemically investigated to elucidate the methodology for repeated batch fermentations for the production of xylitol from xylose. Flocculation always occurred after the late exponential phase of growth in the culture medium and buffer, but did not occur during the exponential phase. The flocculation was optimal within the narrow pH ranges of pH 3.0-5.0. The results were consistent with flocculation by the lectin-like model observed for other yeasts, strongly suggesting that flocculation of HY200 occurs through specific interactions between lectin-like proteins and sugar residues, where calcium is required for the interactions. In order to employ this strain for practical use, suitable conditions for hyper production and yield were established in terms of media formulation and culture parameters. Notably, HY200 used xylitol as the sole carbon source, thereby strongly suggesting that this strain might be a superior candidate for xylitol production from xylose. HY200 was tolerant of high xylose concentrations up to 300 g l^(-1) and was unable to readily metabolize the target product xylitol. The rate of xylitol production increased up to 200 g l^(-1) xylose, and the value in specific xylitol productivity was 2.4 times higher than the value observed at 100 g l^(-1) xylose. Relatively high xylitol yield (77%) and productivity (2.57 g l^(-1) h^(-1)) were obtained when 200 g l^(-1) of xylose was supplemented. In repeated batch fermentation, the HY200 cells were precipitated to remove the fermentation broth, and then the supernatants were transferred into purification after the first batch operation. The HY200 cells were reused for the next fermentation with newly fed xylose solution of an equal composition of nitrogen source. At the conditions of 420 rpm, 0.5 vvm, 30℃, repeated batch fermentation supplemented with xylose, yeast extract, bacto peptone, and tryptone solution resulted in 26.32 g l^(-1) dry cell mass and 6.28 g l^(-1) h^(-1) volumetric productivity in the final batch of the six cycles. We obtained maximal result of a 2.08-fold of volumetric xylitol productivity. Cell recycling by flocculation was very fast, convenient, economically beneficial, and thus potentially applicable for industrial scale of xylitol production.
more초록/요약
Xylitol을 생산하는 당내성 균주인 응집성 균주를 답토로부터 분리하였다. 선별된 균주는 18S rRNA 서열 분석을 통하여 동정한 결과, Candida tropicalis HY200 이라고 명명하였다. HY200의 표현형을 살펴보면 배지내에서 성장한 후에 지속적인 응집현상을 보였다. HY200은 정지기에서 쉽게 응집하여 배양배지에서 분리됨을 알아냈다. Xylose로부터 xylitol을 생산하기 위하여 Candida tropicalis HY200의 응집에 대하여 구조적으로 밝히기위해 체계적으로 연구하였고, 세포 재사용을 통한 반복회분식 발효배양에 적용하였다. 응집현상은 효모의 지수성장 말기 후에 배양배지나 완충용액에서 항상 관찰 되었지만, 지수 성장기 동안에는 관찰할 수 없었다. 또한, 세포의 성장상태, 배양 배지의 pH가 HY200의 응집능력에 대한 요인들이었다. 응집현상은 pH 3.0-5.0 사이의 좁은 최적범위을 나타내었다. 이러한 결과들은 다른 효모들에서 관찰할 수 있었던 the lectin-like model의 응집현상과 일치하였고, HY200은 lectin-like 단백질과 세포표면의 당 잔기와의 특이적 반응을 통하여 응집현상이 일어나고, 여기에 칼슘이 관여하고 있다는 것을 알 수 있었다. 실질적인 산업적 생산에 본 균주를 사용하기 위해서, 높은 생산과 수율위한 배지조성과 배양방법 등에 대하여 적합한 조건을 확립하였다. 주목할만한 특성으로서, HY200은 단일 탄소원으로 xylose를 쉽게 이용할 수 있었고, 따라서 xylose로부터 xylitol 생산을 위한 우수한 균주임을 알 수 있었다. HY200은 xylitol 생산에 있어서 xylose 300 g l^(-1) 까지도 당내성을 나타내었다. Xylitol 생산 수율은 200 g l^(-1) 의 xylose 배지까지 증가되었고, 특이적 xylitol 생산성은 xylose 100 g l^(-1) 보다 2.4 배나 높았다. 최적 배양조건하에서, 실제로, 200 g l^(-1) xylose에서 77%의 높은 수율과 2.57 g l^(-1) h^(-1) 의 생산성을 얻었다. 반복회분식 배양에서, HY200은 쉽게 배양배지에서 분리되어 침전되었고, 상등액은 첫번째 배양이 끝난 후에 정제과정으로 전달되었다. HY200은 새로운 xylose 용액과 동일한 조성의 질소원을 첨가하여 다음 배양에 재사용되었다. 420 rpm, 0.5 vvm, 30℃의 조건에서 반복회분식 배양에 xylose, yeast extract, bacto peptone과 tryptone을 공급하여, 6번의 반복 배양을 진행하였고, 마지막 배양에서는 26.32 g l^(-1) 의 건조균체량과 6.28 g l^(-1) h^(-1)의 생산성을 얻을 수 있었다. 본 연구에서는 반복회분식 배양공정의 최고의 결과로 xyliol 생산성을 회분식 배양보다 2.08배 증가시켰다. 응집현상을 통한 세포의 재사용은 빠르고, 편리하며, 경제적이고, 산업적인 xylitol 생산에 있어서 적용할 수 있는 잠재성이 뛰어난 공정으로 사료된다.
more목차
Contents
List of Tables = ⅰ
List of Figures = ⅱ
Abstract = ⅷ
Chapter Ⅰ. General Introduction = 1
1. What is a xylitol = 1
2. Processes for Xylitol Production = 8
3. Production of Xylitol by Yeasts = 12
4. Industrial Production of Xylitol = 27
5. Flocculation of Yeast = 33
6. Aims of the Study = 41
Chapter Ⅱ. Screening and Characterization of a Flocculent Yeast, Candida sp. HY200 for the Xylitol Production = 42
1. Introduction = 42
2. Materials and methods = 44
2.1. Enzymes and Chemicals = 44
2.2. Culture media = 44
2.3. Screening and Culture conditions = 45
2.4. Strain Identification = 45
2.5. Cloning and sequence analysis of xylose reductase = 46
2.6. Measurement of flocculation ability = 47
2.7. Analyses = 47
3. Results and discussions = 49
3.1. Screening of Novel Flocculent Yeast = 49
3.2. Identification of HY200 = 57
3.3. Sequencing of xylose reductase for HY200 = 63
3.4. Characterization of flocculation for HY200 = 65
3.4.1. Effect of growth phase and cell density on cell flocculation = 65
3.4.2. Effect of pH and temperature on cell flocculation = 67
3.4.3. Effect of inorganic ions on cell flocculation = 73
4. Summary = 82
Chapter Ⅲ. Xylitol Production by Batch Fermentation with Candida tropicalis HY200 = 84
1. INTRODUCTION = 84
2. MATERIALS AND METHODS = 87
2.1. Strain = 87
2.2. Culture Conditions = 87
2.3. Analyses = 88
3. RESULTS AND DISCUSSION = 89
3.1. Media Formulation for Xylitol Production = 89
3.1.1. Effect of carbon sources = 89
3.1.2. Effect of initial xylose concentration = 95
3.1.3. Effect of nitrogen sources = 98
3.2. Optimization of Culture Conditions = 104
3.2.1. Effect of inoculum = 104
3.2.2. Effect of aeration rate = 104
3.2.3. Effect of agitation = 109
3.2.4. Effect of temperature and pH = 109
3.3. Xylitol production by batch fermentation = 111
4. Summary = 115
Chapter Ⅳ. Repeated Batch Fermentation for the Production of Xylitol from D-Xylose with a Flocculent Yeast, Candida tropicalis HY200 = 117
1. INTRODUCTION = 117
2. MATERIALS AND METHODS = 120
2.1. Microorganism = 120
2.2. Culture media = 120
2.3. Culture conditions for repeated batch fermentation with cell recycle by flocculation = 121
2.4. Measurement of flocculation ability = 122
2.5. Analyses = 123
3. RESULTS AND DISCUSSION = 124
3.1. Fed-batch fermentation = 124
3.2. Process of repeated batch fermentation = 126
3.2.1. Effect of repeated batch with/without washing cells = 129
3.2.2. Effect of nitrogen source for flocculation = 131
3.2.3. Improved productivity of xylitol production = 135
3.3. Repeated batch fermentation for xylitol production = 136
4. Summary = 146
Chapter Ⅴ. Conclusion and further Study = 147
Chapter Ⅵ. References = 150
국문요약 = 164
