Immobilization of the Hyperthermophilic Archaeon Thermococcus onnurineus Using Amine-coated Silica Material for H₂ Production

배승섭,나정걸,이성목,강성균,이현숙,이정현,김태완,Bae, Seung Seob,Na, Jeong Geol,Lee, Sung-Mok,Kang, Sung Gyun,Lee, Hyun Sook,Lee, Jung-Hyun,Kim, Tae Wan The Korean Society for Microbiology and Biotechnol 2015 한국미생물·생명공학회지 Vol.43 No.3

초고온성 고세균 Thermococcus onnurineus NA1은 개미산, 일산화탄소, 또는 전분 등을 이용해서 수소를 생산하는 것으로 알려져 있다. 본 연구에서는 T. onnurineus NA1의 고정화 세포를 이용한 수소생산을 고찰하였다. 고정화 실험결과, T. onnurineus NA1은 표면에 아민기가 코팅된 규조토 담체에 정전기적 인력에 의해 효과적으로 고정화되었고, 1 g의 담체에 고정화 될 수 있는 최대 세포의 양은 71.7 mg-dcw로 확인되었다. 고정화 세포를 이용한 세 번의 반복회분식 배양을 통해 개미산으로부터 수소생산 특성을 고찰하였고, 그 결과 배양이 반복됨에 따라 고정화 세포 농도의 증가에 기인하여 초기수소생산속도가 2.3 에서 4.0 mmol l⁻¹ h⁻¹로 상당량 증가됨이 관찰되었다. 따라서, T. onnurineus NA1의 고정화세포 시스템은 수소생산을 위한 좋은 대안이 될 수 있을 것으로 사료된다. 본 연구는 초고온성 고세균의 고정화세포를 수소생산에 적용한 첫 번째 사례이다. Previously we reported that the hyperthermophilic archaeon, Thermococcus onnurineus NA1 is capable of producing hydrogen (H₂) from formate, CO or starch. In this study, we describe the immobilization of T. onnurineus NA1 as an alternative means of H₂ production. Amine-coated silica particles were effective in immobilizing T. onnurineus NA1 by electrostatic interaction, showing a maximum cell adsorption capacity of 71.7 mg-dried cells per g of particle. In three cycles of repeated-batch cultivation using sodium formate as the sole energy source, immobilized cells showed reproducible H₂ production with a considerable increase in the initial production rate from 2.3 to 4.0 mmol l⁻¹ h⁻¹, mainly due to the increase in the immobilized cell concentration as the batch culture was repeated. Thus, the immobilized-cell system of T. onnurineus NA1 was demonstrated to be feasible for H₂ production. This study is the first example of immobilized cells of hyperthermophilic archaea being used for the production of H₂.

Characterization of Thermostable Deblocking Aminopeptidases of Archaeon Thermococcus onnurineus NA1 by Proteomic and Biochemical Approaches

이열균,임선희,정영호,김승일 한국미생물학회 2012 The journal of microbiology Vol.50 No.5

Thermococcus onnurineus NA1 is a hyperthermophilic archaeon that grows optimally at >80°C. The deblocking aminopeptidase (DAP) (TNA1-DAP1) encoded in Ton_1032 of T. onnurineus NA1 is considered a major DAP. However,four genes encoding putative DAP have been identified from a genomic analysis of T. onnurineus NA1. A proteomic analysis revealed that all four DAPs were differentially induced in YPS culture medium and, particularly, two DAPs (TNA1-DAP1 and TNA1-DAP2) were dominantly expressed in T. onnurineus NA1. The biochemical properties and enzyme activity of DAPs induced in an E. coli expression system suggested that the two major DAPs play complementary roles in T. onnurineus NA1.

Immobilization of the Hyperthermophilic Archaeon Thermococcus onnurineus Using Amine-coated Silica Material for H2 Production

( Seungseob Bae ),( Jeonggeol Na ),( Sungmok Lee ),( Sunggyun Kang ),( Hyunsook Lee ),( Junghyun Lee ),( Taewan Kim ) 한국미생물생명공학회(구 한국산업미생물학회) 2015 한국미생물·생명공학회지 Vol.43 No.3

Previously we reported that the hyperthermophilic archaeon, Thermococcus onnurineus NA1 is capable of producing hydrogen (H2) from formate, CO or starch. In this study, we describe the immobilization of T. onnurineus NA1 as an alternative means of H2 production. Amine-coated silica particles were effective in immobilizing T. onnurineus NA1 by electrostatic interaction, showing a maximum cell adsorption capacity of 71.7 mg-dried cells per g of particle. In three cycles of repeated-batch cultivation using sodium formate as the sole energy source, immobilized cells showed reproducible H2 production with a considerable increase in the initial production rate from 2.3 to 4.0 mmol l-1 h-1, mainly due to the increase in the immobilized cell concentration as the batch culture was repeated. Thus, the immobilized-cell system of T. onnurineus NA1 was demonstrated to be feasible for H2 production. This study is the first example of immobilized cells of hyperthermophilic archaea being used for the production of H2.

Proteomic Insights into Sulfur Metabolism in the Hydrogen-Producing Hyperthermophilic Archaeon <i>Thermococcus onnurineus</i> NA1

Moon, Yoon-Jung,Kwon, Joseph,Yun, Sung-Ho,Lim, Hye Li,Kim, Jonghyun,Kim, Soo Jung,Kang, Sung Gyun,Lee, Jung-Hyun,Kim, Seung Il,Chung, Young-Ho MDPI 2015 INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES Vol.16 No.5

<P>The hyperthermophilic archaeon <I>Thermococcus onnurineus</I> NA1 has been shown to produce H<SUB>2</SUB> when using CO, formate, or starch as a growth substrate. This strain can also utilize elemental sulfur as a terminal electron acceptor for heterotrophic growth. To gain insight into sulfur metabolism, the proteome of <I>T</I>. <I>onnurineus</I> NA1 cells grown under sulfur culture conditions was quantified and compared with those grown under H<SUB>2</SUB>-evolving substrate culture conditions. Using label-free nano-UPLC-MS<SUP>E</SUP>-based comparative proteomic analysis, approximately 38.4% of the total identified proteome (589 proteins) was found to be significantly up-regulated (≥1.5-fold) under sulfur culture conditions. Many of these proteins were functionally associated with carbon fixation, Fe–S cluster biogenesis, ATP synthesis, sulfur reduction, protein glycosylation, protein translocation, and formate oxidation. Based on the abundances of the identified proteins in this and other genomic studies, the pathways associated with reductive sulfur metabolism, H<SUB>2</SUB>-metabolism, and oxidative stress defense were proposed. The results also revealed markedly lower expression levels of enzymes involved in the sulfur assimilation pathway, as well as cysteine desulfurase, under sulfur culture condition. The present results provide the first global atlas of proteome changes triggered by sulfur, and may facilitate an understanding of how hyperthermophilic archaea adapt to sulfur-rich, extreme environments.</P>

초고온성 고세균 Thermococcus onnurineus NA1에 의한 수소생산

김옥선,나정걸,김해진,이영우,김미선 한국수소및신에너지학회 2011 한국수소 및 신에너지학회논문집 Vol.22 No.5

A hyperthermophilic archaeon, Thermococcus onnurineus NA1 was studied to investigate its fermentation characteristics using various carbon sources including formate, maltose and carbon monoxide during the anaerobic batch cultivation at 80℃. Formate was the best carbon source for cell growth and hydrogen production among others. In the batch culture on formate, it was found that the cell concentration increased exponentially by 12 hrs of culture, after which the cell growth and formate consumption was retarded. Hydrogen production was continued more than 24 hrs although the cell growth was ceased at 18 hrs. Hydrogen production rate was directly correlated with the cell growth and formate degradation up to 18 hrs, and the average hydrogen production yield was 1.05 mole-H2/mole-formate. Cell growth and hydrogen production were optimized at the initial pH 6-7, while inhibited at the initial pH lower than 5 and higher than 9.

Enhancement of the hydrogen productivity in microbial water gas shift reaction by <i>Thermococcus onnurineus</i> NA1 using a pressurized bioreactor

Kim, Min-Sik,Fitriana, Hana Nur,Kim, Tae Wan,Kang, Sung Gyun,Jeon, Sang Goo,Chung, Soo Hyun,Park, Gwon Woo,Na, Jeong-Geol Elsevier 2017 INTERNATIONAL JOURNAL OF HYDROGEN ENERGY - Vol.42 No.45

<P><B>Abstract</B></P> <P>Here, we developed a pressurized bioreactor system that increase carbon monoxide (CO) transfer efficiency in order to enhance the hydrogen productivity in the microbial water gas shift reaction by <I>Thermococcus onnurineus</I> NA1. The effects of CO pressure on the hydrogen production rate, CO consumption rate and the cell growth were investigated using small scale stainless steel bottles at various CO partial pressures. It was found that CO solubility increased by applying pressure can affect hydrogen production positively as long as the increased toxicity of CO is endurable to cells. The hydrogen productivity increased to some extent with CO pressure, but decreased drastically at the pressure higher than 4 bar. On the other hand, the effect of pressure itself on the cell's activity was not as significant as that of CO solubility increase. In the experiments at various system pressures with identical CO partial pressure of 1 bar, more than 80% of the cell activity remains even at total pressure of 10 bar. Also, it was important to determine the appropriate time to increase pressure for preventing excess CO in the reactor. Based on these results, a fermentation strategy for the pressurized system was designed and applied to a 5 L bioreactor with the continuous supply of the gas containing 60% CO. When the pressure was introduced to the bioreactor up to 4 bar at CO limitation condition, the unprecedented high productivity (360 mmol L<SUP>−1</SUP> h<SUP>−1</SUP>) could be obtained.</P> <P><B>Highlights</B></P> <P> <UL> <LI> CO gas solubility increased by pressurizing can be beneficial to H<SUB>2</SUB> production. </LI> <LI> The impact of pressure per se was marginal. </LI> <LI> CO toxicity impacts were reduced by applying pressure at mass transfer limitation condition. </LI> <LI> When the pressure was introduced to the bioreactor, the unprecedented high productivity could be obtained. </LI> </UL> </P>

Determination of Optimal Dissolved CO Concentration for Bioreactor Operation Using Thermococcus onnurineus NA1 Strains

Nulee JANG,Muhammad YASIN,Yeseul JEONG,Shinyoung PARK,Yeubin LEE,In Seop CHANG,Hyunsoo KANG 한국생물공학회 2015 한국생물공학회 학술대회 Vol.2015 No.10

Oxygen-mediated growth enhancement of an obligate anaerobic archaeon Thermococcus onnurineus NA1

Seong Hyuk Lee,Hwan Youn,Sung Gyun Kang,Hyun Sook Lee 한국미생물학회 2019 The journal of microbiology Vol.57 No.2

Thermococcus onnurineus NA1, an obligate anaerobic hyperthermophilic archaeon, showed variable oxygen (O2) sensitivity depending on the types of substrate employed as an energy source. Unexpectedly, the culture with yeast extract as a sole energy source showed enhanced growth by 2-fold in the presence of O2. Genome-wide transcriptome analysis revealed the upregulation of several antioxidant-related genes encoding thioredoxin peroxidase (TON_0862), rubrerythrin (TON_0864), rubrerythrin-related protein (TON_0873), NAD(P)H rubredoxin oxidoreductase (TON_0865), or thioredoxin reductase (TON_1603), which can couple the detoxification of reactive oxygen species with the regeneration of NAD(P)+ from NAD(P)H. We present a plausible mechanism by which O2 serves to maintain the intracellular redox balance. This study demonstrates an unusual strategy of an obligate anaerobe underlying O2-mediated growth enhancement despite not having heme-based or cytochrome-type proteins.

해양바이오수소개발 사업의 상업생산을 위한 예비경제성평가

박세헌,유영돈,강성균 한국해양과학기술원 2016 Ocean and Polar Research Vol.38 No.3

This project sought to conduct an economic feasibility study regarding the commercial production of bio-hydrogen by the marine hyperthermophilic archaeon, Thermococcus onnurineus NA1 using carbon monoxide-containing industrial off-gas. We carried out the economic evaluation of the biohydrogen production process using the raw material of steel mill by-product gas. The process parameter was as follows: H2 production rate was 5.6 L/L/h; the conversion of carbon monoxide was 60.7%. This project established an evaluation criterion for about 10,000 tonne/year. Inflation factors were considered as 3%. The operating costs were recalculated based on prices in 2014. The total investment required for development was covered 30% by capital and 70% by a loan. The operation cost for the 0.5-year test and integration, and the cost for the first three months in the 50% production period were considered as the working capital in the cost estimation. The costs required for the rental of office space, facilities, and other related costs from the construction through to full-scale production periods were considered as continuing expenses. Materials, energy, waste disposal and other charges were considered as the operating cost of the development system. Depreciation, tax, maintenance and repair, insurance, labor, interest rate charges, general and administrative costs, lubrication and miscellaneous expenses were also calculated. The hydrogen price was set at US$ 4.15/kg for the economic evaluation. As a result, the process was considered to be economical with the payback period of 6.3 years, NPV of 18 billion Won and IRR of 26.7%.

Dimethyl sulfoxide reduction by a hyperhermophilic archaeon Thermococcus onnurineus NA1 via a cysteine-cystine redox shuttle

Ae Ran Choi,Min-Sik Kim,Sung Gyun Kang,Hyun Sook Lee 한국미생물학회 2016 The journal of microbiology Vol.54 No.1

A variety of microbes grow by respiration with dimethyl sulfoxide (DMSO) as an electron acceptor, and several distinct DMSO respiratory systems, consisting of electron carriers and a terminal DMSO reductase, have been characterized. The heterotrophic growth of a hyperthermophilic archaeon Thermococcus onnurineus NA1 was enhanced by the addition of DMSO, but the archaeon was not capable of reducing DMSO to DMS directly using a DMSO reductase. Instead, the archaeon reduced DMSO via a cysteine-cystine redox shuttle through a mechanism whereby cystine is microbially reduced to cysteine, which is then reoxidized by DMSO reduction. A thioredoxin reductase-protein disulfide oxidoreductase redox couple was identified to have intracellular cystine-reducing activity, permitting recycle of cysteine. This study presents the first example of DMSO reduction via an electron shuttle. Several Thermococcales species also exhibited enhanced growth coupled with DMSO reduction, probably by disposing of excess reducing power rather than conserving energy.