KR102430707B1 - Thermococcus onnurineus WTF-350T and methods of hydrogen production by using thereof - Google Patents

Abstract

The present invention relates to a Thermococcus onnurinus WTF-350T strain and a hydrogen production method using the same. The WTF-350T strain according to the present invention can produce hydrogen using formic acid, and cell density ( OD ₆₀₀ ) increased by 32% and was 16% higher at the maximum hydrogen production rate. Therefore, the strain according to the present invention is applicable to producing hydrogen at a high level from formic acid, and producing high yield of hydrogen at low cost.

Description

Thermococcus onnurineus WTF-350T strain and hydrogen production method using the same {Thermococcus onnurineus WTF-350T and methods of hydrogen production by using thereof}

The present invention relates to a Thermococcus onnurinus WTF-350T strain and a hydrogen production method using the same.

The biological hydrogen production method using microorganisms has advantages in that there is no need to create high temperature and high pressure conditions by inputting additional energy, and carbon monoxide is not included in the generated gas. This biological hydrogen production method can be largely divided into using photosynthetic microorganisms and non-photosynthetic microorganisms (mainly anaerobic microorganisms). However, in the former case, high-concentration culture technology of photosynthetic bacteria using light as an energy source has not yet been sufficiently developed, and conventional photosynthetic bacteria have a disadvantage in that substrate inhibition is severe in the presence of a high partial pressure of substrate. In addition, they have a problem that hydrogen generation performance can be sustained only in the presence of light. Therefore, attempts to produce hydrogen using microorganisms capable of producing hydrogen using organic carbon are continuously being made. The present inventors revealed that hydrogen can be produced from carbon monoxide using a strain of the genus Thermococcus through Korean Patent Application No. 10-2010-7013071 (published on June 23, 2011). In addition, a patent was registered as 10-1534483 for the WTC155T strain with high hydrogen production capacity from carbon monoxide. Entered Korea in 2019-7019704. The present inventors developed a strain with 15% higher hydrogen production capacity than the WTC-156T strain through continuous improvement and completed the present invention.

It is an object of the present invention to provide a Thermococcus onnurineus WTF-350T ( Thermococcus onnurineus WTF-350T) strain.

Another object of the present invention is to provide a method for producing hydrogen by supplying formic acid to the Thermococcus onnurineus WTF-350T ( Thermococcus onnurineus WTF-350T) strain.

In order to achieve the above object, the first aspect of the present invention provides a Thermococcus onnurineus WTF-350T ( Thermococcus onnurineus WTF-350T, Accession No.: KCTC 14212BP) strain.

More specifically, the Thermococcus onnurinus WTF-350T strain is a strain capable of producing hydrogen at a high concentration by using formic acid as a substrate.

A second aspect of the present invention comprises the steps of: (a) supplying and culturing formic acid to a strain of Thermococcus onnurinus WTF-350T; And (b) provides a hydrogen production method comprising the step of separating hydrogen from the culture.

The strain according to the present invention increased the cell density (OD ₆₀₀ ) by 32% compared to the WTF-156T strain in the method for producing hydrogen using formic acid, and was 16% higher in the maximum hydrogen production rate. This means that the hydrogen productivity by the strain according to the present invention is significantly increased. The strain according to the present invention is applicable to high-yield hydrogen production at low cost.

1 is a comparison of the results of batch culture in a bioreactor in which a wild-type strain, a WTF-156T strain, and a WTF-350T strain were provided with 400 mM formate as a substrate. (a), growth curves in wild-type (black symbols), WTF-156 (grey symbols) and WTF-350 (white symbols); (b), hydrogen productivity; (c) The concentrations of residual formic acid and hydrogen were compared. The pH was adjusted to 6.1-6.2 using 2N HCl containing 3.5% NaCl as pH adjuster.
Figure 2 compares the growth of the wild type strain (Wild type) and the WTF-350T strain in formate substrate conditions containing oxygen. After inoculating each strain in a sealed test tube, 20% oxygen was supplemented in each head space. The cell densities of the non-inoculated control (blank) and wild-type strains cultured in the presence of oxygen were measured to be 0.004 ± 0.002 and 0.011 ± 0.001, respectively, and the cell density of the WTF-350T strain was measured to be 0.096 ± 0.007. Cell growth was measured for absorbance at 600 nm (OD ₆₀₀ ) after 12 hours of incubation at 80 degrees Celsius. All experiments were independently repeated three times.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention in more detail, and it is understood by those of ordinary skill in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. it will be self-evident

Example 1 Development of a strain according to the present invention and hydrogen production using the same

strain and medium

T. onnurineus strain NA1 (KCTC 10859) was isolated from a deep-sea hydrothermal vent in PACMANUS Field 31, Papua New Guinea - Australia - Canada. This strain is fertilized medium 1 (MM1) [Kim, YJ et al . Formate-driven growth coupled with H ₂ production. Nature 467, 352-355 (2010); Sokolova, TG et al . The first evidence of anaerobic CO oxidation coupled with H ₂ production by a hyperthermophilic archaeon isolated from a deep-sea hydrothermal vent. Extremophiles . 8, 317-323 (2004)]. For mutagenesis, single base substitutions were performed as previously described [Jung, HC et al , Adaptive evolution of a hyperthermophilic archaeon pinpoints a formate transporter as a critical factor for the growth enhancement on formate. Scientific Reports. 7, 6124 (2017)] was prepared by homologous recombination. The formic acid-adapted strain was cultured in serum vials in MM1 medium containing 1 g L ^-1 of yeast extract and 147 mM sodium formate at 80 °C, and 2% of each culture was replaced with fresh medium every 15 hours. was moved to

T. onnurineus Incubation of NA1

All cultures were performed in a 3 L bioreactor (CNA, Daejeon, South Korea) containing 1-1.5 L of MM1 medium supplemented with an appropriate amount of yeast extract and sodium formate. Stirring speed, temperature and pH were maintained at 500 rpm, 80° C. and 6.2, respectively.

Anaerobic culture was performed by flowing N ₂ gas into the reactor at least 30 minutes before inoculation. For pH-fixed batch culture, strains were cultured in 1.5 L of MM1 medium containing 4 or 10 g L ⁻¹ yeast extract and 400 mM sodium formate while adjusting the pH with 2 or 5N HCl in 3.5% NaCl. For the pH fixed formic acid supply system, 5 N formic acid containing 3.5% NaCl was used for pH adjustment and formic acid supply. Batch culture was started in 1 L MM1 medium containing 10 g L ^-1 and 400 mM sodium formate, pH adjusted with 5N formic acid containing 3.5% NaCl.

After 3 hours of culture, 0.02 Lh ^-1 of 10X MM1 medium containing 100 g L ^-1 yeast extract was supplied using a peristaltic pump. For continuous culture, 1x medium containing 10 gL ^-1 yeast extract was supplied according to a dilution rate after 7 hours, and an appropriate amount of 10 N formic acid was supplied to supplement formic acid. The volume of the reactor was kept constant while the bioreactor was operating by discarding more than 1 L in a recycling manner.

Analysis method

Cell growth was measured at optical density at 600 nm (OD ₆₀₀ ) with a biophotometer and UV visible-spectrum spectrometer (Eppendorf, Hamburg, Germany). The concentration of biomass was determined as previously described [Lim, JK et al, Appl Environ Microbiol. 2012 Oct; 78(20): pp.7393`7397)] was determined from the correlation between dry cell weight (DCW) and <sub>OD600</sub> . The concentration of formic acid was analyzed using HPLC (YL instrument Co, Anyang, South Korea) with a UV detector and RSpak KC-811 column (Shodex, Tokyo, Japan) at 1.0 ml min <sup>-1</sup> with H <sub>3</sub> PO <sub>4</sub> as the mobile phase. . The concentrations of H <sub>2</sub> and CO <sub>2</sub> in the headspace of the bioreactor were measured using a Molsieve5 Acolumn (Supelco, Bellefonte, PA, USA), a Porapak N column (Supelco), a thermal conductivity detector (TCD), a flame ionization detector. detector; FID) and YL6100 GC gas chromatography equipped with a methanizer (YL instrument Co., Anyang, South Korea). Argon was used as the carrier gas, and the flow rate was 30 ml/min. The total volume of the discharge gas of the bioreactor was measured using a wet gasmeter (Shinagawa, Tokyo, Japan) at 1 atm. The actual Gibbs free energy change (ΔGs) for formic acid-dependent H <sub>2</sub> production was calculated as the standard Gibbs free energy (ΔG˚) value and the actual measured reactant and product concentrations and partial pressures using the Nernst equation. . ΔG˚ at different temperatures is determined by Amend and Shock's paper [Amend and Shock, Energetics of overall metabolic reactions of thermophilic and hyperthermophilic Archaea and bacteria. FEMS Microbiol Rev. 2001;25(2):175-243 (2001)]. A more detailed procedure for the thermodynamic calculation was as previously reported (Lim, JK et al, Appl Environ Microbiol. 2012 Oct; 78(20): pp.7393`7397).

result

In order to develop a strain with increased hydrogen production capacity from formic acid, strain WTF-156T adapted in formic acid medium was further adapted by transferring to formic acid medium in a serum vial for an additional 200 times. Genomic analysis of WTF-350T found 16 additional mutations in addition to 11 mutations found in WTF-156T, which are summarized in Table 1 below.

[Table 1] Mutations observed in the WTF 350T genome

^a All mutations were confirmed by PCR and Sanger sequencing.

It was found that the 104th serine amino acid of formate transferase (Ton_1573; formate transporter) was substituted with arginine. In addition to formate transferase, additional mutations were found in the fdh2-mfh2-mnh2 gene. These two mutations are thought to be representative genomic changes that explain the increase in hydrogen production capacity found in WTF-350T. However, these two mutations as well as other mutations may be important, and their effects should be further studied.

The WTF-350T strain showed a maximum hydrogen production capacity of 16% higher than that of the WTF-156T strain ( FIG. 1 , Table 2 ), and showed a high final cell concentration (OD ₆₀₀ ) of 32% ( FIG. 2 ). This shows that the strain according to the present invention has significantly improved the performance of the strain through additional adaptive mutations.

[Table 2] Kinetic analysis of wild-type and WTF-156T and WTF-350T

The kinetic coefficients were calculated using the data from FIG. 1 .

μ _max , specific proliferation rate; r _max , maximum hydrogen productivity; q _max , the maximum specific hydrogen productivity.

^a Biomass productivity was determined by dividing the total yield from 11 hours to 13 hours for the wild-type strain and from 2 hours to 4 hours for the WTF-156T strain and WTF-350T strain.

^b Hydrogen productivity was determined by dividing total production by time.

The Thermococcus onnurineus WTF-350T strain was deposited on the bioresource seton 2020.06.12, and was given an accession number as KCTC 14212BP.

Claims (3)

Thermococcus onnurineus ( Thermococcus onnurineus ) KCTC 14212BP strain. According to claim 1, Thermococcus onnurineus capable of producing hydrogen from formic acid ( Thermococcus onnurineus ) KCTC 14212BP strain. (a) culturing by supplying formic acid to the strain of claim 1; and
(b) hydrogen production method comprising the step of separating hydrogen from the culture.

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