초록 ▼

본 사업은 해양초고온 고세균을 이용하여 경제적인 바이오수소 생산기술을 개발하고자 하였음. 이를 위해 다음과 같은 연구들을 수행함.

1. 초고온 고세균의 수소생산 기작규명: 오믹스 분석을 통하여 수소생산 관련 전사조절자의 특성 규명, 단백질체 및 대사체 분석, 기질별 수소생산 메카니즘 규명, 및 수소생산 대사회로 작성함.

2. 수소생산 우수균주개발: 오믹스 분석결과를 바탕으로 수소생산 관련 효소들이 과발현 되거나 일산화탄소에 적응기작된 다수의 우수균주들을 개발함. 그 중 WTC156T 우수 균주는 야생형 대비

본 사업은 해양초고온 고세균을 이용하여 경제적인 바이오수소 생산기술을 개발하고자 하였음. 이를 위해 다음과 같은 연구들을 수행함.

1. 초고온 고세균의 수소생산 기작규명: 오믹스 분석을 통하여 수소생산 관련 전사조절자의 특성 규명, 단백질체 및 대사체 분석, 기질별 수소생산 메카니즘 규명, 및 수소생산 대사회로 작성함.

2. 수소생산 우수균주개발: 오믹스 분석결과를 바탕으로 수소생산 관련 효소들이 과발현 되거나 일산화탄소에 적응기작된 다수의 우수균주들을 개발함. 그 중 WTC156T 우수 균주는 야생형 대비 수소생산능이 2배 이상 증가함.

3. 가스발효 최적 반응기 개발 및 수소생산성 최적화: 가스발효에 적합한 마이크로버블러 반응기 및 airlift 반응기를 개발하였고, 연속공정 최적화를 통해 수소생산성을 극대화함. 최대수소생산성 250mmol/L/h로 세계최고 수준 달성함.

4. 원료물질 확보 및 공급시스템 구축: 경제적인 원료물질 확보위해 제철소 및 석탄가스화 플랜트 보유기관 등과의 연구협력 추진함. 최종적으로 6차년도에 고등기술연구원의 석탄가스화 플랜트와 연구협력 체결함. 고등기술연구원의 시설을 보강하여 석탄가스화 합성가스의 바이오리액터로의 공급시스템 구축함.

5. 파일럿플랜트 구축 및 바이오수소 시범생산: 연속배양이 가능한 1톤 배양기 규모 파일럿 플랜트 구축함. 일산화탄소 이용하여 최대 수소생산속도 165 mmol/L/h 달성하였고, 최종적으로 석탄가스화 합성가스를 공급하면서 바이오수소 시범생산을 성공함.

6. 경제성 분석: 현재의 수소생산성 (250mmol/L/h)을 토대로 연간 1만톤의 수소생산 규모를 가정하여 경제성 분석을 수행함. 자본회수기간 6.3년, 순현재가치 180억원, 내부수익률 26.7%로 경제성이 높을 것으로 예상됨.

Abstract ▼

IV. Results

(1) Understanding the mechanism for H₂ production of T. onnurineus NA1: a study of characterization of transcriptional regulator
- Screening of putative transcriptional regulators and choose eight-hypothetical transcriptional regulators: TON_0037, TON_0282, TON_0318,

IV. Results

(1) Understanding the mechanism for H₂ production of T. onnurineus NA1: a study of characterization of transcriptional regulator
- Screening of putative transcriptional regulators and choose eight-hypothetical transcriptional regulators: TON_0037, TON_0282, TON_0318, TON_0332, TON_0548, TON_0836, TON_1016 and TON_1797)
- Screening of possible regulator protein which was strongly bound to target promoter DNAs: TrmBL2 and TON_0332

(2) Understanding the mechanism for H₂ production of T. onnurineus NA1: a transcriptime study

- Construction and sequencing of stranded RNA-seq library from NA1 transcriptome
- Construction and sequencing of differential RNA-seq library from NA1 transcriptome
- Genome analysis of NA1 from the sequencing data of stranded RNA-seq library and differential RNA-seq
- Identification of genes from the analysis of transcriptome expression pattern in YPS, CO, or Formate condition

(3) Understanding the mechanism for H₂ production of T. onnurineus NA1: a proteome/metabolom study

- A comparative proteomic analysis of T. onnurineus NA1 cells grown on CO, formate, starch, and sulfur
- A comprehensive metabolomic analysis of the intracellular carbon assimilation in T. onnurineus NA1 cells grown on different substrates (formate, CO, starch)
- Development of fractionation method for analysis of membrane proteome and secretome
- Membrane proteome analysis of T. onnurineus NA1 by 1DE-LCQ- MS/MS analysis and interpretation of their metabolic characterization
- Establishment of proteomic platform technology for comprehensive understanding of hyperthermophilic archaea

(4) Development of engineered strains of T. onnurineus NA1 with higher activity for H₂ evolution on carbon monoxide

- Development of genetic tool-box and construction of mutants
- Development of genetically engineered strain of T. onnurineus NA1, MC01, which showed 30% higher activity for H₂ evolution than wild type
- Development of genetically engineered strains of T. onnurineus NA1 in which the regulational protein was over-expressed, showing higher activity for H₂ evolution than wild type
- Development of genetically engineered strains of T. onnurineus NA1 via optimization of cell membrane space
- Development of CO-specific adapted strain of T. onnurineus NA1, WTC156T, which showed 3-fold higher activity for H₂ evolution than wild type
- Characterization for cell growth and H2 production of NA1 strains with higher H₂ evolution activity than wild type and confirmation of process stability from long-term continuous operation over 1 month

(5) Development of optimal reactors for gas-fermentation and process optimization for H₂ production

- Characterization of cell growth and H2 production by continuous culture of NA1 in CSTR : NA1 showed a stable H₂ production rate (HPR) of 316.5 mmol/L/h at dilution rate of 0.025 h^-1. In case of artificial LDG gas (CO:H₂:CO₂:N₂ = 57:2:15:26, v/v), HPR was 327.5mmol/L/h at gas flow rate of 0.4 vvm without any inhibitions by LDG gas
- Development of spiral microbubble generator (SMG) equipped with circulation pump (0.5–3 HP)
- Development of 20L and 30L scale microbubble reactors internally installed with SMG (I-CMR)
- Development of 20L and 30L scale microbubble reactors externally connected with SMG (E-CMR) for convenient maintenance
- Continuous culture of NA1 in E-CMR : In 20 L E-CMR, HPR was 304.8 ± 4.1 mmol/L/h at QCO of 0.2 vvm at ambient pressure. HPR could be increased to 449.0 ± 11.3 mmol/L/h, which corresponded to 2.41 Nm³ per day, at QCO of 0.4 vvm at pressure of 2 bar. In addition, using 100 L E-CMR(2 bar, QCO 0.2 vvm), we could obtain HPR of 267.5 ± 14.0 mmol/L/h (5.99 ± 0.31 L/L/h), which corresponded to 5.75 Nm³ per day. In case of continuous culture supplying LDG gas at 0.2 vvm in 20 L E-CMR, HPR was 223.3 mmol/L/h(5.23 ± 0.09 L/L/h), CO conversion (XCO) was 76.5% and H₂ content was 32.9 ± 0.6%. To our knowledge, these showed the best hydrogen production efficiency.
- Development of airlift bio-reactor: Internal-loop type airlift bioreactor was developed and its operability was improved by the optimization of internals. The bioreactor was scaled-up into 1 ton-scale pilot plant after some modifications and its performance was confirmed.
- Development of cell immobilization technology: We synthesized a magnetic nanoparticle having affinity to T. onnurineus NA1 and it was applied for the cell immobilization. NA1 cell shows negative charges on its surface and a particle with positive charge can attract the cells.

(6) Enhancement of gas-liquid mass transfer efficiency of carbon monoxide

- Determination of intrinsic kinetic parameters (K_s and KM_i) of T. onnurineus NA1 wild-type, recombinant(MC01 and MC02) and adaptive strain WTC156T. WTC156T showed much higher consumption rate of carbon monoxide than the other strains - Hollow fiber membrane bioreactor (HFMBR) system for improvement of CO mass transfer in bioconversion: High k_La of 155.16 h^-1 was achieved by increasing A_s/V_L to 0.62 cm^-1 under lower transmembrane pressure of 5.4 psi.
- Development of microbubble generator: In bio-reactor, microbubble can be generated by transferring supersaturated CO in a pressurized vessel (solver) into bio-reactor. This strategy could produce microbubbles without high shear forces required in the conventional microbubble generator. The amount of CO transfer could be controlled by circulation rate of medium and the pressure of solver.
- Design of a metallic sparger of which surface was controlled by the deposition of hydrophilic functional group via PECVD method to obtain smaller size bubbles

(7) Securing raw materials and development of supplying system

- Investigation of H₂ produciton of NA1 from steel mill by-product gas : As a result of fed-batch cultivation of T. onnurineus NA1 on artificial gas of BFG (Blast Furnace Gas) steel mill by-product gas, we can obtain H2 productivity of 17.4 L/L/day with CO conversion of 100%. As a result of fed-batch cultivation of T. onnurineus NA1 on artificial gas of LDG (Lintz Donawiz Gas) steel mill by-product gas, we can obtain H2 productivity of 32.4 L/L/day with CO conversion of 77.7%. As a result of fed-batch cultivation of T. onnurineus NA1 on real LDG gas, we can obtain H2 productivity of 64.3 mmol/L/h with CO conversion of 77.7%
- Investigation of H₂ produciton of NA1 from syngas from gasification of coal or biomass: As a result of batch cultivation of genetically engineered strain of T.onnurineus NA1, MC01, on artificial gas of syngas from coal gasification (H₂:35.48%, CO:35.2%, CO₂:28.3%, CH₄:1.02%), we can obtain maximum H₂ production rate of 58.2 mmol/L/h at agitation speed of 600 rpm. As a result of batch cultivation of genetically engineered strain of T. onnurineus NA1, MC01, on syngas from coal gasification (CO content 40%), we can obtain maximum H₂ production rate of 49.0 mmol/L/h at agitation speed of 600 rpm - Construction of supplying system of raw material into bioreactor: Supplying system of syngas obtained from coal gasification or 100% CO gas into bio-hydrogen production reactor was developed with a supplying capacity of maximum 50 Nm³/h at pressure of up to 3 bar.
- Investigation of H₂ produciton of NA1 from organic waste matters: Yeast extract, cysteine, Vitamin can be 100% replaced with effluents from the anaerobic digestion. Additionally, other organic wastes like food leachate, cattle manure and sewage sludge can replace the nutrients although their scope is rather limited.

(8) Construction of ton-scale pilot plant and demonstration of bio-hydrogen production

- Construction of pilot plant : We constructed pilot plant for bio-hydrogen production consisting of one gas-lift type reactor (total voume 1 ton), two feed tanks (total volume 2.5 ton), PSA (pressure swing adsorption) unit for H₂ purification and H₂ storage tank (9.9 bar, 200 L)
- Selection of strain with the highest H₂ evolving activity among many engineered strains of NA1 : We Screened the three strains showing higher H₂ evolving activity among 9 candidates in serum bottle. The three strains were WTC156T, WTC 290T, △frh::HMG, KS0510. From the kinetic analysis of batch cultivations of three screened strains in 3L fermentor, the WTC156T strain was finally selected as a strain to be applied to pilot plant.
- Characterization of cell growth and H₂ production of WTC156T strain on coal-gasified syngas in lab scale fermentor : As a result of batch cultivation of CO specific adapted strain of T. onnurineus NA1, WTC156T, on syngas from coal gasification (CO content 40%), we can obtain maximum H₂ production rate of 81.4 mmol/L/h at agitation speed of 600 rpm without any inhibition
- Batch cultivation of NA1 on carbon monoxide in pilot plant: As a result of batch cultivation of WTC156T strain on carbon monoxide at ambinent pressure in pilot plant, the maximum cell concentration was OD600 of 1.8 and H₂ production rate was 82.6 mmol/L/h. As a result of batch cultivation of WTC156T strain on carbon monoxide at 1 atm in pilot plant, the maximum cell concentration was OD600 of 1.8 and H₂ production rate was 165.6 mmol/L/h. It was considered that the enhancement of H2 production rate was due to the increase of CO solubility at elevated pressure, showing that pressure can be the critical operational parameter.
- Demonstration of continuous bio-hydrogen production from coal-gasified syngas in pilot plant: We demonstrate the bio-hydrogen production in pilot plant by the continuous cultivation of WTC156T on syngas from coal gasification. Dilution rate was 0.12 h-1, gas flow rate was 0.06-0.08 vvm, and the composition of syngas was H₂ 25.6%, CO 40.3%, CO₂ 8.4%, CH₄ 0.7%. In early stage of cultivation, H₂ production rate (HPR) and CO conversion (Xco) were reached up to 114.3 mmol/L/h and 71.1%, respectively. Foam-breaking technology which do not inhibit cell activity should be developed for stable operation.
- Design of process flow diagram for bio-hydrogen production using coal-gasified syngas or steel mill by-product gas : We developed the concept design of the demo-scale bio-hydrogen production system including process flow diagram (PFD), Heat & mass balance, P&ID, and reactor design.

(9) Economical evaluation

- Economic assessment of microbial WGS process using steel mill by-product gas (LDG): Various scenarios for utilization of steel mill gas were derived for economic assessment of the microbial water gas shift process by T. onnurineus NA1. It shows that the process developed in this project is economically feasible compared than that of the conventional process (electricity generation by gas turbines), however it is necessary to validate the process at the demonstration scale in order to resolve doubts regarding bioprocess.
- We carried out economical evaluation of the bio-hydrogen production process using raw material of steel mill by-product gas. The process parameter was as follows: H₂ production rate was 5.6 L/L/h; the operational pressure was 1 atm; the annual H₂ production was 10,000 ton.
- As a result, the process is considered to be economical with the payback periods of 6.3 years, NPV of 18 billion Won and IRR of 26.7%