[보고서]당화효소 및 효모의 추가 투입 없이 동시당화 발효에 의한 맥주 폐효모액으로부터 바이오 에탄올 생산

당화효소 및 효모의 추가 투입 없이 동시당화 발효에 의한 맥주 폐효모액으로부터 바이오 에탄올 생산
Bio-ethanol production by the simultaneous saccharification and fermentation process using the waste from beer fermentation broth without additional enzyme and microbial cells 원문보기

보고서 정보
주관연구기관	경북대학교 KyungPook National University
보고서유형	최종보고서
발행국가	대한민국
언어	한국어
발행년월	2014-05
과제시작연도	2013
주관부처	미래창조과학부 Ministry of Science, ICT and Future Planning
등록번호	TRKO201500004156
과제고유번호	1345199037
사업명	중견연구자지원
DB 구축일자	2015-05-23
키워드	바이오 에탄올.연료용 에탄올.맥주 폐 효모액.대체 에너지.산업 폐기물.고온 단기 발효.동시 당화 발효.고농도 균체 발효.에탄올 생산성.bioethanol.fuel ethanol.waste from beer fermentation broth.alternative energy.industrial waste.short term thermo-fermentation.simultaneous saccharification and fermentation.fermentation with high cell concentration.productivity.
DOI	https://doi.org/10.23000/TRKO201500004156

초록 ▼

연구의 목적 및 내용
(1) 산업 폐기물인 맥주 폐효모액를 이용한 친환경적 경제적 바이오 에탄올 생산. (2) 맥주 폐효모액에 존재하는 맥아 유래 당화효소와 맥주 폐 효모를 이용한 동시 당화발효 mechanism 확립. (3) 당화효소의 최적 온도와 에탄올 생산용 효모전세포 상시효소를 이용한 고온 바이오에탄올 생산 mechanism 확립. (4) 고온 동시 당화 발효공정에 의한 바이오 에탄올 생산 공정 시간의 획기적 단축. (5) 연속식 고농도 맥주 폐효모 에탄올 발효 기술을 이용한 에탄올 생산성 증가.
연구결과
(1) one-pot 동시당화발효 공정을 통한 맥주 폐효모액으로부터의 바이오에탄올 생산
교반 조건과 정치 조건에서 맥주폐효모액의 상등액은 각각 103.8g/L, 91.5g/L을 생산하였다. 연속적인 one-pot 동시당화발효 공정은 첫번째 단계(25-67℃)에서 102.5g/L, 두번째 단계(67℃)에서 219g/L의 바이오에탄올을 생산할 수 있었다.
(2) 맥주 폐효모액에 존재하는 효소의 동정 및 부분적 정제
SDS-PAGE와 LC–MS/MS Q-TOF 분석을 이용하여 맥아 유래 가수분해 효소와 맥주 폐효모액의 당화 및 발효 효소의 동정을 진행하였다.
(3) 바이오에탄올 생산을 위한 효모 무세포 효소 시스템의 개발
단일 효모 세포에 기반한 정제된 무세포 효소 시스템을 개발하였고 특성을 밝혀 40℃에서 바이오에탄올 생산.
(4) 효소 재사용과 반복된 batch 공정을 위한 캡슐화 무세포 효소 시스템의 개발
단일효모 세포 유래 cofactor(보조인자)와 발효 효소를 칼슘 알지네이트 캡슐에 고정화함으로써 캡슐화된 무세포 효소 시스템이 개발되었고 15번의 연속적인 batch 바이오에탄올 생산.
연구결과의 활용계획
(1) WBFB(맥주폐효모액)로부터 바이오에탄올 생산
(2) 고농도 효모균 유래의 무세포효소 시스템을 동시당화발효과정에 적용하여 바이오 에탄올을 생산해내는 세계적인 원천기술 개발
(3) 연료용 바이오 에탄올 생산 외에도 연구결과를 알코올 생산, 식품발효, 대체에너지 생산 등에 광범위하게 응용
(4) 미래 산업에서 요구되는 BT와 ET 융합 기술에 대한 전문 인력 양성

Abstract ▼

Purpose&contents
(1) Economic and eco-friendly production of bioethanol from the industrial waste, the waste from beer fermentation broth (WBFB) (2) Establishment of the mechanism of the simultaneous fermentation and saccharification using the enzymes from malt and yeast from WBFB (3) Establishment of the mechanism of bioethanol production at high temperature using saccharification enzymes and whole cell constitutive enzyme from yeast (4) Remarkable reduction in production time of bioethanol by simultaneous saccharification and fermentation thermo-process (5) High productivity of bioethanol by the continuous high cell concentration process.
Result
(1) Bioethanol production from WBFB through SSF one-pot process.
Bare supernatant of WBFB produced 103.8g/L and 91.5g/L ethanol in shaking and static conditions, respectively through SSF. A consecutive two phase one-pot SSF operation revealed bioethanol production of 102.5g/L in first phase (25-67℃) and 219g/L in second phase (67℃).
(2) Identification and partial purification of enzymes in WBFB.
We identified malt hydrolyzing and yeast glycolytic and fermentation enzymes in WBFB through SDS-PAGE and LC–MS/MS Q-TOF analyses analysis.
(3) Development of yeast cell free enzyme system for bioethanol production.
A purified cell free enzyme system based on single yeast cells was developed, characterized and successfully evaluated for bioethanol production at 40℃.
(4) Development of encapsulated cell free enzyme system for enzymes reusability and repeated batch operation
An encapsulated cell free enzyme system consisting of fermentation enzymes from yeast cells along with cofactors confined in calcium alginate capsules was developed and successfully evaluated for bioethanol production for 15 consecutive batches.
Expected Contribution
(1) Worldwide milestone for the bioethanol production from WBFB.
(2) Worldwide original technology for the bioethanol production using simultaneous saccharification and fermentation thermo-reactions with yeast cells of high concentration.
(3) Worldwide application of the current research results to the other alcohols production, food fermentation and alternative energy production.
(4) Training specialists of the next generation in the merging technology of BT and ET.

목차 Contents

핵심연구사업 최종보고서(평가용) ... 1
목 차 ... 3
연구계획 요약문 ... 4
연구결과 요약문 ... 5
한글요약문 ... 5
SUMMARY ... 6
연구내용 및 결과 ... 7
1. 연구개발과제의 개요 ... 7
2. 국내외 기술개발 현황 ... 8
3. 연구수행 내용 및 결과 ... 9
4. 목표달성도 및 관련분야에의 기여도 ... 71
5. 연구결과의 활용계획 ... 92
6. 연구과정에서 수집한 해외과학기술정보 ... 92
7. 주관연구책임자 대표적 연구실적 ... 93
8. 참고문헌 ... 94
9. 연구성과 ... 100
10. 기타사항 ... 106
끝페이지 ... 112

표/그림 (37)

표 Structural formula of carboxymethyl cellulose (CMC) and cellulose.
표 WBFB (old and new) after every step of purification was processed for endoglucanase activity by using CMC as substrate in the presence of citrate buffer pH 4.8 with incubation temperature of 50°C for 30 min
표 Enzyme extract obtained after every purification step (a supernatant; b (NH4)2SO4saturation;c dialysis and d acetone precipitation) was analyzed by SDS-PAGE to determine the molecular weight of expected malt derived hydrolyzing enzymes in WBFB.
표 One-pot consecutive batch strategy for ethanol production in 850 mL double jacketed bioreactor with 850 mL working volume under static conditions. The WBFB supernatant was used at 67°C in the first phase of cultivation, followed by an additional second phase at 25°C with 3% solid residue. Arrow means one-pot addition of WBFB solid residue.
표 Cell wall hydrolyzing activity of enzyme extracts from WBFB. Samples were incubated in the presence of 1% CMC as substrate in citrate buffer at different pHs (3.0-6.0) and at 50°C for 30 min. The activity of cell wall hydrolyzing enzymes at pH 4.5 was taken as 100%
표 Cell wall hydrolyzing activity of enzyme extract from WBFB. Samples were incubated in the presence of 1% CMC as substrate in citrate buffer at pH 4.5 and 30°C-80°C for 30 min. The activity of cell wall hydrolyzing enzymes at 50°C was taken as 100%
표 Thermal stability of cell wall hydrolyzing enzyme. Samples were preincubated at 30°C–80°C for 1 h and the catalytic activity was measured under standard assay conditions. The activity of cell wall hydrolyzing enzymes at 30°C after different intervals was taken as 100%.
표 Microscopic pictures of S. cerevisiae in chemically defined medium with enzyme extract taken at 25°C–70°C after intervals of a 15; b 30; c 45; d 60; e 75; f 90; g 105; and h 120 min.
표 Morphology of yeast cells after different intervals of beating; A (0 min), B (1 min), C (2.5 min), D (5 min), E (7.5 min), F (10 min) and G (15 min).
표 Microscopy of cell-free solution obtained after bead beating, centrifugation and filtration.
표 Colony forming units of each sample after various intervals of bead beating measured using YM agar plates incubated at 30ºC for 48 hr.
표 Total protein concentration in a medium was found to increase as a result of mechanical disruption of the yeast cell membrane by bead beating with 600 μm glass beads at lower temperature.
표 SDS PAGE analysis of yeast cell lysate produced after bead beating for different intervals of time A; 0 min, B; 1 min, C; 2.5 min, D; 5 min, E; 7.5 min, F; 10 min and G; 15 min along with standard. All 12 fermentation enzymes are shown in the figure (HK; Hexokinase, PGI; Phosphoglucose Isomerase, PFK; Phosphofructo Kinase, Ald; Aldolase, TPI; Triphosphate Isomerase, GAPDH; Glyceraldehydes Phosphate Dehydrogenase, PGK; Phosphoglycerate Kinase, PGM; Phosphoglycerate Mutase, Eno; Enolase, PK; Pyruvate Kinase, PDC; Puruvate Decarboxylase and ADH; Alcohol Dehydrogenase).
표 Cell-free enzyme extract obtained after bead beating of WBFB containing yeast was evaluated for bioethanol production at different temperatures (30, 40, 50 and 60ºC).
표 Evaluation of bioethanol production using different concentrations of cell-free enzyme extract of WBFB yeast (1, 2, 3 and 4 ml) at the optimum temperature (40ºC).
표 The kinetics profile of (A) starch hydrolysis, (B) glucose production, (C) residual glucose accumulation, and (D) bio-ethanol production by the encapsulated and bare reconstituted cell-free enzyme systems during SSF course. The experiments were performed at 45°C and pH 7.0 using 1% starch with agitation (150 rpm) for cell-free enzyme concentration hours.
표 SDS-PAGE analysis of the encapsulated reconstituted cell-free enzyme system after 15 consecutive SSF batches. The “cell-free enzyme” lane contains the cell-free enzymes after 15 consecutive batches, whereas the positive control lane contains the initial cell-free enzyme solution.
표 Enzyme extract obtained after every purification step (a supernatant; b (NH4)2SO4saturation;c dialysis and d acetone precipitation) was analyzed by SDS-PAGE to determine the molecular weight of expected malt derived hydrolyzing enzymes in WBFB.
표 Cell wall hydrolyzing activity of enzyme extracts from WBFB. Samples were incubated in the presence of 1% CMC as substrate in citrate buffer at different pHs (3.0-6.0) and at 50°C for 30 min. The activity of cell wall hydrolyzing enzymes at pH 4.5 was taken as 100%
표 Cell wall hydrolyzing activity of enzyme extract from WBFB. Samples were incubated in the presence of 1% CMC as substrate in citrate buffer at pH 4.5 and 30°C-80°C for 30 min. The activity of cell wall hydrolyzing enzymes at 50°C was taken as 100%
표 Cell-free enzyme system analyzed by LC–MS/MS (Q-TOF) using the mascot algorithm to identify yeast cell enzymes.
표 Thermal stability of cell wall hydrolyzing enzyme. Samples were preincubated at 30°C–80°C for 1 h and the catalytic activity was measured under standard assay conditions. The activity of cell wall hydrolyzing enzymes at 30°C after different intervals was taken as 100%.
표 Microscopic pictures of S. cerevisiae in chemically defined medium with enzyme extract taken at 25°C–70°C after intervals of a 15; b 30; c 45; d 60; e 75; f 90; g 105; and h 120 min.
표 SDS-PAGE analysis of yeast cell lysate produced after bead beating for different intervals of time: A, -min; B, 1 min; C, 2.5 min; D, 5 min; E, 7.5min; F, 10min; and G, 15 min. A standart is also shown. All ten fermentation enzymes are shown in the figure (PGM, Phosphoglycerate Mutase; TPI, Triphosphate Isomerase; ADH, Alcohol Dehydrogenase; Ald, Aldolase; Eno, Enolase; GAPDH, Glyceraldehyde Phosphate Dehydrogenase; HK, Hexokinase; PK Pyruvate Kinase; PGI, Phosphoglucose Isomerase; and PDC, Pyruvate Decarboxylase).
표 The effects of different temperatures on cell-free bio-ethanol production were evaluated using a cell-free enzyme solution (1.56 mg/mL) and 1% glucose solution with agitation (150 rpm) and pH 7.0 after 8 h of fermentation.
표 Cell-free bio-ethanol production at different pH values using a cell-free enzyme solution (1.56 mg/mL) and 1% glucose solution. The reaction mixtures were incubated at 40◦C with agitation (150 rpm) for 8 h
표 The effects of different concentrations of cell-free enzyme solution on bioethanol production were evaluated using 3% and 5% initial glucose at 40℃ with agitation (150rpm) at pH 7.0 for 8h.
표 Effect of glucose concentration on bio-ethanol production using a cell-free enzyme system. Fermentation was performed using a cell-free enzyme concentration of 1.56 mg/mL with agitation (150 rpm) at 40◦C and pH 7.0 for 8 h.
표 Comparative analysis of a cell-free enzyme system and conventional fermentation system for bio-ethanol production at five different temperatures with agitation (150 rpm) at pH 7.0 for 8 h using same amount of live cells and cell-free enzyme concentration (1.56 mg/mL) obtained from equal volume of yeast pre-culture.
표 Synergistic effect of thermal stress and cell wall hydrolyzing enzymes on yeast cell morphology after: (a) (1h), (b) (2h), (c) (3h), (d) (4h), (e) (5h), (f) (6h) and (g) (7 h). After 5 h, 3% solid sediments that were mostly dominated by yeast cells were added.
표 Illustration of the total protein and glucose concentration in WBFB supernatant before and during fermentation, and the bio-ethanol production profile in one-pot consecutive batches under 50rpm using 850mL of WBFB supernatant as the first phase in an 850mL double jacketed jar fermenter at 67℃, followed by a second phase of cultivation at 67℃ with 3% WBFB solid residue. The arrow indicates the second phase of fermentation and error bars indicate the standard deviation from triplicate values.
표 Three different classes of enzymes including saccharification enzymes from malt and glycolytic and fermentation enzymes from yeast were identified in WBFB supernatant using SDS-PAGE.
표 FE-SEM photographs of precipitates formed in 50mL vials with a 50mL working volume of unheated WBFB supernatant (a and b) and after heat treatment of the WBFB supernatant at 67◦C during the course of bio-ethanol production (c and d). The vials were placed in a beaker containing water and positioned in an 850 mL double jacketed bioreactor connected to a water circulating bath.
표 FTIR spectra of the freeze– dried WBFB supernatant and sediments formed after heat treatment of the WBFB supernatant at 67◦C in 50 mL vials with a 50 mL working volume during the course of bio-ethanol production. The vials were placed in a beaker containing water and positioned in an 850 mL double jacketed bioreactor connected to a water circulating bath.
표 Several saccharification enzymes from malt and glycolytic and fermentation enzymes from yeast were identified in WBFB supernatant by SDS-PAGE analysis using a 15% polyacrylamide gradient gel under reducing conditions.
표 WBFB supernatant was analyzed by LC–MS/MS (Q-TOF) using the mascot algorithm to identify yeast cell proteins present in WBFB.
표 Comparative analysis of the SSF and SHF process describing the overall economics of SSF for bio-ethanol production.

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