해조류 우뭇가사리 (Gelidium amansii)의 분리당화발효를 이용한 바이오에탄올의 생산

라채훈(Chae Hun Ra),이현준(Hyeon Jun Lee),신명교(Myung Kyo Shin),김성구(Sung-Koo Kim) 한국생물공학회 2013 KSBB Journal Vol.28 No.5

The seaweed, Gelidium amansii, was fermented to produce bioethanol. Optimal pretreatment condition was determined as 94 mM H₂SO₄ and 8% (w/v) seaweed slurry at 121℃ for 60 min. The mono sugars of 40.4 g/L with 67% of conversion from total carbohydrate of 60.6 g/L with 80 g dw/L G. amansii slurry were obtained by thermal acid hydrolysis pretreatment and enzymatic saccharification. G. amansii hydrolysate was used as the substrate for ethanol production by Kluyveromyces marxianus KCTC 7150 and Candida tropicalis KCTC 7212 using 5L fermentor. The ethanol productions by K. marxianus KCTC 7150 and C. tropicalis KCTC 7212 were 17.8 g/L with YEtOH of 0.48 at 120 h and 19.3 g/L with YEtOH of 0.50 at 120 h, respectively.

발효, 세포배양, 생물공정 탄소원 조성 조절을 이용한 Azotobacter indicus var. myxogenes L3로부터 PS-7 생산 최적화

라채훈 ( Chae Hun Ra ),김기명 ( Ki Myong Kim ),허필우 ( Pil Woo Hoe ),이성재 ( Sung Jae Lee ),김성구 ( Sung Koo Kim ) 한국미생물생명공학회 2008 한국미생물·생명공학회지 Vol.36 No.1

해조류 바이오매스를 이용한 바이오에탄올 생산기술

라채훈(Chae Hun Ra),선우인영(In Young Sunwoo),김성구(Sung-Koo Kim) 한국생명과학회 2016 생명과학회지 Vol.26 No.8

해조류는 성장이 빠르고, 낮은 경작지 사용, 높은 이산화탄소 흡수 및 식량자원과 경쟁하지 않는 장점이 있다. 따라서 리그닌이 없는 해조류 사용은 바이오에탄올 생산을 위한 3세대 바이오매스로 주목받고 있다. 산 촉매 열가수분해 전처리법은 해조류로부터 높은 단당을 획득할 수 있는 경제적인 방법 중 하나이다. 고온 전처리 조건들에서 3,6-anhydrogalactoe는 저해물질인 HMF로 전환되는데, 이 저해물질은 세포 성장과 에탄올 생산을 저해한다. 따라서 바이오에탄올을 생산하기 위해 해조류의 탄수화물을 분해할 때는 높은 단당 수율과 낮은 저해물질 생성을 하는 효과적인 전처리 방법이 필요하다. 혼합 당을 이용한 에탄올 발효의 효율을 향상시키기 위해, 고농도 당에 순치한 효모는 혼합 당의 사용을 통해 해조류를 이용한 바이오 에탄올의 생산을 가능하게 한다. Seaweed has high growth rate, low land usage, high CO2 absorption and no competition for food resources. Therefore, the use of lignin-free seaweed as a raw material is arising as a third generation biomass for bioethanol production. Various pretreatment techniques have been introduced to enhance the overall hydrolysis yield, and can be categorized into physical, chemical, biological, enzymatic or a combination. Thermal acid hydrolysis pretreatment is one of the most popular methods to attain high sugar yields from seaweed biomass for economic reasons. At thermal acid hydrolysis conditions, the 3,6-anhydro-galactose (AHG) from biomass could be converted to 5-hydroxymethylfurfural (HMF), which might inhibit the cell growth and decrease ethanol production. AHG is prone to decomposition into HMF, due to its acid-labile character, and subsequently into weak acids such as levulinic acid and formic acid. These inhibitors can retard yeast growth and reduce ethanol productivity during fermentation. Thus, the carbohydrates in seaweed require effective treatment methods to obtain a high concentration of monosaccharides and a low concentration of inhibitor HMF for ethanol fermentation. The efficiency of bioethanol production from the seaweed biomass hydrolysate is assessed by separate hydrolysis and fermentation (SHF). To improve the efficiency of the ethanol fermentation of mixed monosaccharides, the adaptation of yeast to high concentration of sugar could make simultaneous utilization of mixed monosaccharides for the production of ethanol from seaweed.

홍조류(Kappaphycus alvarezii)의 동시 당화 발효를 이용한 바이오에탄올의 생산

라채훈 ( Chae Hun Ra ),김성구 ( Sung Koo Kim ) 한국미생물생명공학회(구 한국산업미생물학회) 2016 한국미생물·생명공학회지 Vol.44 No.2

해조류 중 홍조류인 K. alvarezii로부터 동시 당화 발효(SSF)를 위한 효소 당화 및 균 배양 온도를 검토하고, 기존의 동시 당화 발효(SSF) 를 개선하기 위해 2단계 동시 당화발효(SSF)를 수행하였다. 효소 당화와 균 성장 온도를 고려 하였을 때 동시 당화 발효(SSF)에 적용하는 배양 온도는 40℃를 선택하여 실험을 진행하였다. 비순치 효모(wild type)와 고농도 갈락토오스에 순치한 효모(adapted yeast to galactose)를 이용한 동시 당화 발효(SSF)를 실시한 결과 발효 156시간에 9.1 g/l의 에탄올 수율(YEtOH) 0.24와 10.2 g/l의 에탄올 수율(YEtOH) 0.27을 나타내었다. 이러한 기존의 동시 당화 발효(SSF)를 개선한 2단계 동시 당화 발효(SSF)는 에탄올 생산 수율이 0.27에서 0.35로 27.5% 증가하였으며, 에탄올 발효 시간도 156시간에서 96시간으로 61.5% 감소하였다. 이러한 연구결과는 해양 바이오매스인 해조류로부터 바이오연료 생산과정에 있어 기초적인 정보를 제공할 것이다. Thermal acid hydrolysis pretreatment of Kappaphycus alvarezii was carried out with 12% (w/v) seaweed slurry and 180 mM H2SO4 at 140°C for 5 min. Utility of the thermotolerant yeast Kluyveromyces marxianus KCTC7150 was evaluated with respect to cell growth and ethanol fermentation at 40°C was close to optimal for enzymatic hydrolysis. This could lead to the integration of both the saccharification and fermentation processes. The levels of ethanol production by simultaneous saccharification and fermentation (SSF) with non-adapted and adapted K. marxianus KCTC7150 were 9.1 g/l with an ethanol yield (YEtOH) of 0.24 and 10.2 g/l with an ethanol yield (YEtOH) of 0.27 at 156 h, respectively. The two-phase SSF process was employed in this study to improve the efficiency of ethanol fermentation. Adapted K. marxianus KCTC7150 using the two-phase SSF process produced 13.5 g/l with an ethanol yield (YEtOH) of 0.35 at 96 h. Development of the two-phase SSF process could enhance the overall ethanol fermentation yields of the seaweed K. alvarezii.

Evaluation of Optimal Condition for Recombinant Bacterial Ghost Vaccine Production with Four Different Antigens of Streptococcus iniae-enolase, GAPDH, sagA, piaA

Chae-Hun Ra(라채훈),Yeong-Jin Kim(김영진),Chang-Woo Son(손창우),Dae-Young Jung(정대영),Sung-Koo Kim(김성구) 한국생명과학회 2009 생명과학회지 Vol.19 No.7

본 연구는 5-L 발효기를 이용하여 재조합 고스트 박테리아(E. coli DH5α / pHCE-InaN-(enolase, GAPDH, sagA or piaA)-ghost 37 SDM) 백신의 산업화를 위해 탄소원 공급조건, 교반속도, 산소공급 조건 등의 최적 배양조건과 고스트 박테리아 발현 유도를 위한 온도조절 시점과 그에 따른 발현효율 최적화를 조사하기 위해 수행하였다. 각각 다른 4종의 항원 유전자를 보유한 고스트 박테리아를 LB 배지를 이용하여 배양한 결과 모두 1 g/l glucose, 300 rpm, 1 vvm에서 최대 균주 성장을 나타내었다. 고스트 박테리아 생성 효율의 경우 초기 대수증식기(OD600=1.0)에서 고스트 발현을 유도했을 때 각각 최대효율인 99.99%를 나타내었으나 중기 대수증식기(OD600=2.0)와 말기 대수증식기(OD600=3.0)에서는 고스트 박테리아 생성이 낮은 효율을 나타내었다. 또한 SDS-PAGE 와 western blot를 이용하여 각각 다른 4종의 항원 단백질 발현 여부를 확인한 결과 enolase (78 kDa), GAPDH (67 kDa), sagA (2 6kDa), piaA (26 kDa) 에서 항원 단백질 band를 확인할 수 있었다. 따라서 본 연구결과 확립된 배양 조건과 발현효율 최적화 조건은 연쇄구균증 질병에 대해 E. coli를 이용한 고스트 박테리아 백신이 양식 산업에 있어 상업적으로 유용한 백신의 최적생산을 위해 사용 될 수 있을 것으로 사료된다. A vector harboring double cassettes; a heterologous gene expression cassette of pHCE-InaN-antigen and a ghost formation cassette of pλPR-cI-E lysis 37 SDM was constructed and introduced to E. coli DH5α. For the production of a bacterial ghost vaccine, bacterial ghosts from E. coli / Streptococcus iniae with four different types of antigens - enolase, GAPDH, sagA and piaA - were produced by the optimization of fermentation parameters such as a glucose concentration of 1 g/l, agitation of 300 rpm and aeration of 1 vvm. Efficiency of ghost bacteria formation was evaluated with cultures of OD600=1.0, 2.0 and 3.0. The efficiency of the ghost bacteria formation was 99.54, 99.67, 99.99 and 99.99% with inductions at OD600=3.0, 1.0, 2.0 and 1.0 for E. coli / S. iniae antigens enolase, piaA, GAPDH and sagA, respectively. Ghost bacteria as a vaccine was harvested by centrifugation. The antigen protein expressions were analyzed by SDS-PAGE and western blot analysis, and the molecular weights of the enolase, piaA, GAPDH and sagA were 78, 26, 67 and 26 kDa, respectively. The molecular weights of the expressed antigens were consistent with theoretical sizes obtained from the amino acid sequences.

Optimization of the Medium Composition for Heteropolysaccharide-7 Production by Beijerinckia indica L3 Using Response Surface Methodology

Chae Hun Ra(라채훈),Ki Myong Kim(김기명),Pil Woo Hoe(허필우),Mi Ran Choi(최미란),Sung-Koo Kim(김성구) 한국생명과학회 2008 생명과학회지 Vol.18 No.2

본 연구는 플라스크 배양을 통해 B. indica L3 균주를 가지고 PS-7을 생산할 수 있는 최적 조건을 조사하기 위해서 수행하였다. RSM을 통한 배지의 최적화 실험에서는 whey lactose, glucose, ammonium nitrate 세 가지의 독립변수의 조성에 따라 배양 72시간에 해당하는 DCW, PS-7, 그리고 점도의 변화에 대한 결과를 관찰하였다. 결정 계수(R square)의 값은 PS-7생산량, DCW, 점성에서 각각 0.72, 0.64, 0.85로 신뢰성 있는 값을 획득하였다. Whey lactose와 glucose의 조합에 따른 PS-7 생산에 미치는 효과를 살펴보면 whey lactose (2%), glucose (1%)에서 최적의 PS-7 생산을 나타내었다. 결론적으로 glucose에 whey lactose를 기반으로 하는 배지 첨가와 C/N 비율이 PS-7의 생산에 큰 영향을 미침을 알 수 있었다. The production of heteropolysaccharide-7 (PS-7) by Beijerinckia indica (B. indica L3) was evaluated in shaker flask culture. The medium optimization was studied using response surface methodology (RSM). A five-level three-factor central composite design was employed to determine the maximum PS-7 yield at optimum levels for whey lactose, glucose and ammonium nitrate contents. The validity of the model could be determined by the regression coefficient, R². The values of R² were 0.72, 0.64 and 0.85 in PS-7, DCW and viscosity, respectively. The optimal medium combinations of whey lactose, glucose and ammonium nitrate concentrations on the PS-7 production were whey lactose (2%), glucose (1%) and ammonium nitrate 5 mM, respectively. The result indicated that PS-7 production was affected significantly by the addition of glucose to whey lactose based on medium and C/N ratio.

Beijerinckia indica L3 fermentation for the effective production of heteropolysaccahride-7 in various carbon sources and compositions in whey medium

Ki Myong Kim(김기명),Chae Hun Ra(라채훈),Sung-Koo Kim(김성구) 한국생물공학회 2005 한국생물공학회 학술대회 Vol.2005 No.10

Lactic Acid Bacteria (LAB)와 5-L 발효기를 이용한 γ-Aminobutyric Acid 생산기술 개발

김나연 ( Na Yeon Kim ),김지민 ( Ji Min Kim ),라채훈 ( Chae Hun Ra ) 한국미생물 · 생명공학회 2021 한국미생물·생명공학회지 Vol.49 No.4

유산균 L. plantarum SGL058과 L. lactis SGL027로부터 OFAT 방법을 이용하여 탄소원 및 질소원의 종류와 농도에 대해 실험을 진행하였다. 또한 TLC 분석방법을 통해 GABA 생성능력이 우수한 균주 L. plantarum SGL058과 L. lactis SGL027를 선정하였다. 두 균주를 이용하여 각각 5-L 발효기에서 546.7 μg/ml (L. plantarum SGL058)과 404.6 μg/ml (L. lactis SGL027)의 GABA를 생산하였다. 이러한 연구결과는 유산균을 이용한 GABA 생산과정에 있어서 유용한 정보를 제공할 것으로 판단된다. This study aimed to optimize gamma-aminobutyric acid (GABA) production by employing five strains of lactic acid bacteria (LAB) that were capable of high cell growth and GABA production using a modified synthetic medium. GABA production in the strains was qualitatively confirmed via detection of colored spots using thin layer chromatography. Lactobacillus plantarum SGL058 and Lactococcus lactis SGL027 were selected as the suitable strains for GABA production. The conditions of the carbon and nitrogen sources were determined as 5 g/l glucose (L. plantarum SGL058), 5 g/l lactose (L. lactis SGL027), 10 g/l yeast extract (L. plantarum SGL058), and 20 g/l yeast extract (L. lactis SGL027) for GABA production. The cell growth, monitored by optical density at 600 nm, was 5.93 for L. plantarum SGL058. This value was higher than the 3.04 produced by L. lactis SGL027 at 36 h using a 5-L fermenter. The highest concentration of GABA produced was 546.7 μg/ml by L. plantarum SGL058 and 404.6 μg/ml by L. lactis SGL027, representing a GABA conversion efficiency of (%, w/w) of 4.0% and 3.4%, respectively. The fermentation profiles of L. plantarum SGL058 and L. lactis SGL027 provide a basis for the utilization of LAB in GABA production using a basal synthetic medium.

Eucheuma spinosum으로부터 다양한 효모를 이용한 바이오에탄올 생산

김민지(Min-Ji Kim),김정수(Jung-Soo Kim),라채훈(Chae Hun Ra),김성구(Sung-Koo Kim) 한국생물공학회 2013 KSBB Journal Vol.28 No.5

Ethanol fermentations were performed using separate hydrolysis and fermentation (SHF) processes with monosaccharides from pretreated seaweed, Eucheuma spinosum as the biomass. The pretreatment was carried out with 11% (w/v) seaweed slurry and 150 mM H₂SO₄ at 121℃ for 40 min. Enzyme hydrolysis after H₂SO₄ pretreatment was performed with Celluclast 1.5 L at 45℃ for 24 h. Five % active charcoal were added to hydrolysate to removed 5-hydroxy methylfurfural. Ethanol fermentation with 11% (w/v) seaweed hydrolysate was performed for 72~96 h using Kluyvermyces marxianus, Pichia stipits, Saccharomyces cervisiae and Candida tropicalis. Ethanol concentration was reached to 18 g/L by K. marxianus, 16 g/L by P. stipitis, 15 g/L by S. cerevisiae and 10 g/L by C. tropicalis, respectively. The ethanol yield from total monosugar was obtained 0.50 and ethanol productivity was obtained 0.38 g/L/h by K. marxianus.

홍조류(Eucheuma denticulatum)를 이용한 바이오에탄올 생산

김상원 ( Sang Won Kim ),곽승희 ( Seung Hee Gwak ),라채훈 ( Chae Hun Ra ),김성구 ( Sung-koo Kim ) 한국미생물생명공학회(구 한국산업미생물학회) 2017 한국미생물·생명공학회지 Vol.45 No.4

Hyperthermal acid hydrolysis pretreatment of Eucheuma denticulatum was carried out using 12% (w/v) seaweed slurry and 90 mM H₃PO₄ at 150℃ for 10 min. The use of Candida lusitaniae with adaptive evolu-tion was evaluated for ethanol fermentation. The levels of ethanol production by separate hydrolysis and fermentation (SHF) at 72 h with non-adapted and adapted C. lusitaniae were 10.1 g/l with ethanol yield (Y_EtOH) of 0.23, and 18.1 g/l with YEtOH of 0.45, respectively. Adaptive evolution was employed in this study to improve the efficiency of ethanol fermentation. Development of the SHF process could enhance the overall ethanol fermentation yields of the red seaweed E. denticulatum.