Bioethanol Production from the Waste Product of Salted Undaria pinnatifida Using Laboratory and Pilot Development Unit (PDU) Scale Fermenters

라채훈,강창한,정귀택,김성구 한국생물공학회 2014 Biotechnology and Bioprocess Engineering Vol.19 No.6

The waste product from salted Undaria pinnatifida(sea mustard) processing was fermented to producebioethanol by Saccharomyces cerevisiae KCCM 1129 atlaboratory and pilot development unit (PDU) scales. Thermalacidic hydrolysis of salted U. pinnatifida was conductedwith 75 mM H2SO4 at 121oC for 60 min and the additionof 1.4 KNU/mL Termamyl 120L. A total monosaccharideconcentration of 19.3 g/L and 32.2% conversion from59.9 g/L total carbohydrate using 130 g dw/L saltedU. pinnatifida were achieved. Ethanol fermentations in 5and 500 L fermenters were carried out to produce 8.5 g/Lof ethanol with an ethanol yield (YEtOH) of 0.44 at 24 h and7.9 g/L with YEtOH of 0.41 at 18 h, respectively. Thefermentation time of the PDU-scale reaction was reduceddue to differences in the impeller type and geometry of thefermenters.

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

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.

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.

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.

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.

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.

겉보리의 발아조건 및 혼합발효를 이용한 효소 및 베타글루칸 생산 연구

이세연,라채훈 한국유산균·프로바이오틱스학회 2021 Current Topic in Lactic Acid Bacteria and Probioti Vol.7 No.2

Evaluation of Pretreatment and GABA Production Using Levilactobacillus brevis Fermentation of the Seaweed Saccharina japonica

이세연,라채훈 한국생물공학회 2023 Biotechnology and Bioprocess Engineering Vol.28 No.4

The seaweed has a high content of easily degradable carbohydrates, making it a potential substrate for the production of γ-aminobutyric acid (GABA). In this study, response surface methodology pretreatment and enzymatic saccharification (Es) were conducted on a flask culture of Saccharina japonica seaweed. The optimal hydrolytic conditions were: 10.8% (w/v) slurry content, 0.7% H2SO4, and 121°C for 30 min. Es using enzyme cocktails (Celluclast 1.5 L + Viscozyme L) at 16 U/mL produced 6.26 g/L glucose with an efficiency of 92%. The concentrations of laminarin and fucose (prebiotics) were 10.4 and 0.48 g/L after pretreatment and saccharification, respectively. The suitable monosodium glutamate (MSG) addition was 2% (w/v), and further increase in MSG addition (3–5% (w/v)) had no significant effect on GABA production. The pyridoxal 5′-phosphate (10 μM) addition time of 48–72 h was determined based on the GABA fermentation. Adapted Levilactobacillus brevis KCL010 to high concentrations of mannitol improved the synbiotic fermentation efficiency of S. japonica hydrolysates, further improving the consumption of mixed monosaccharides.

Aspergillus niger를 이용한 곡류발효물의 배양특성 및 α-Glucosidase 활성 저해효과

지수빈,라채훈 한국유산균프로바이오틱스학회 2022 Current Topic in Lactic Acid Bacteria and Probioti Vol.8 No.2

Liquid-state fermentation (LSF) and solid-state fermentation (SSF) were carried out to produce enzymes and β-glucan. The modified synthetic medium composition in liquid-state fermentation (LSF) was determined to be 5% (w/v) whole oat flour, 2% (w/v) tryptone, and 2% (w/v) rice bran. The different humidity conditions of oat by solid-state fermentation were evaluated in terms of maximum production of fungal biomass, amylase, protease, and β-glucan, which were 0.23 mg/g, 7,157.38 U/g, 413.67 U/g, and 8.26% (w/w), respectively, at 60% of humidity condition. Moreover, α-glucosidase inhibition activities of fermented oat with 60% humidity at 48 h were shown to be high 49.08% (w/w). Therefore, liquid- and solid-state fermentation processes were found to enhance the overall fermentation yields of oat to produce enzymes and β-glucan.