일산화탄소를 수소로 전환하는 화학종속영양균의 분리 및 동정

김중래*, 정현옥*, 정규열**, 박성훈*,**

부산대학교 화학공학과*, 부산대학교 환경기술·산업개발 연구센터**

Isolation and Identification of Chemoheterotrophic Bacterium Which Converts Carbon Monoxide to Hydrogen

Jung Rae Kim*, Hyun Ok Jung*, Gyoo Yeol Jung**, Sunghoon Park*,**

Department of Chemical Engineering, Pusan National University*

Institute for Environmental Technology and Industry, Pusan National University**

Introduction

Biological conversion of carbon monoxide to hydrogen by various kinds of microorganisms has been investigated for several decades. Some photoautotrophic bacteria such as Rhodopseudomonas gelatinosa and Rhodospirillum rubrum were reported to convert carbon monoxide to hydrogen.[1, 2] Most prominent strain was Rhodospirillum rubrum, photoautotrophic purple non-sulfur bacterium, which can grow under carbon monoxide as sole carbon and energy source and has the highest maximum specific carbon monoxide conversion rate among the microorganisms reported so far. However, growth rate and cell concentration of this microorganism is relatively low and so volumetric carbon monoxide conversion rate is low. Therefore, some efforts for enhancement of volumetric carbon monoxide conversion rate have been attempted but not successful[3, 4], and so isolation and screening of high-performance microorganism for this purpose is necessary.

In this report, chemoheterotrophic bacterium which converts carbon monoxide to hydrogen was isolated from upflow airlift sludge blanket (UASB) reactor for decolorization of dye wastewater and identified by 16S rDNA cloning and sequencing. In addition, growth and performance for the conversion of carbon monoxide to hydrogen were compared to those of the other reported microorganisms.

Materials and Methods

Enrichment and Isolation of Microorganisms

Carbon-monoxide-utilizing microorganisms were selectively isolated from anaerobic enrichment cultures. About 45 ml of chemoheterotrophic growth medium (described below) was prepared, but with 0.2 g/L of yeast extract and without adding bacto-tryptone, and inoculated 5 ml sample of anaerobic, black sludge from upflow airlift sludge blanket reactor operated for decolorization of dye wastewater in the Laboratory of Biochemical Engineering of Pusan National University. Enrichment cultures were incubated at 30℃ under Ar-CO gas (80:20 mixture) atmosphere. When turbid growth developed, 5 ml of cells was obtained from enrichment culture and transferred into 45 ml of sterile reduced liquid medium prepared as before. After cells had been subcultured under the Ar-CO gas atmosphere, 5 ml of cells was transferred into chemoheterotrophic growth medium consisting of (grams per 1 liter distilled H2O): bacto-tryptone, 20; yeast extract, 10; NH4Cl, 2.7; KH2PO4, 0.5;  MgCl2·6H2O, 0.33; CaCl2·2H2O, 0.05; and NaCl, 0.4. The mineral solution of Pfennig and Lippert [6] and a vitamin solution [7] were added in 9.5 and 8.5 ml amount respectively, to 1 liter of medium. Chemoheterotrophic subculture was incubated at 30℃ under air-CO gas (80:20 mixture) atmosphere.  After cells had been subcultured chemoheterotrophically, a loopful of cells was spread onto sterile solid medium prepared in a petri-dish. Using routine techniques, chemoheterotrophic bacterium was isolated from enrichment culture that grew in strictly dark conditions under air-CO (80:20 mixture) gas.[5]

Growth and Conversion of Carbon Monoxide to Hydrogen

Cells were grown in the chemoheterotrophic growth medium described above for two days and harvested by centrifugation at 8,000 rpm for 5 minutes at 4℃. Cells were then resuspended in Pfennig's hydrogen production medium[3]. At the beginning of  hydrogen production period, cells were adapted under Ar-CO gas (80:20 mixture) for a day and adapted under Ar-CO gas (60:40 mixture) at the next day. After the adaptation, conversion of carbon monoxide to hydrogen was accomplished under pure CO gas (1 atm).

PCR Amplification and Cloning

16S rDNA libraries were constructed by polymerase chain reaction amplification of DNA extracted from the isolated cells, followed by cloning into pCRII (TA cloning kit, Invitrogen, Carlsbad, CA) [8]

Gas Measurements

Carbon monoxide conversion to hydrogen was measured using DS6200 gas chromatograph (Donam Instruments Inc., Seoul) equipped with a molecular sieve 5A column and thermal conductivity detector. Argon was used as the carrier gas.

DNA Sequencing and Analysis

Plasmids containing 16S rDNA inserts were sequenced using an ABI PRISM dye terminator cycle seuencing kit with AmpliTaq DNA polymerase (Protocol P/N 402078 Revision A) and an Applied Biosystems 373 DNA Sequencer (Perkin Elmer, Foster City, CA) Greater than 400 base pairs were sequenced for each insert using single primer extension with the 1492r primer or 27f primer [9].

All sequences were analyzed by the CHECK_CHIMERA and SIMILARITY_RANK programs of the Ribosomal Database Project [10]. The sequences were also analyzed using the BLAST program (National Center for Biotechnology Information) to determine the closest available database sequences.

Results and Discussion

Morphology of Isolated Cell

Morphology of isolated cell was determined by transmission electron microscopy (Fig. 1). Isolated cell had a shape of rod, yellowish color (Fig. 1a), and the ability of sporulation (Fig. 1b).

Fig. 1. Transmission electron microscope of isolated cell. (a) well-grown cell (b) spores.

Growth and Carbon Moxide Conversion to Hydrogen

Isolated cell could grow chemoheterotrophically only. For the purpose of the induction of carbon monoxide conversion system, it was cultivated under air-CO (80:20 mixture) atmosphere. After 48-hour cultivation, stationary phase began and cell concentration reached at OD600 = 3.5 (Fig. 2). Cells in 30 ml of the culture were then harvested and resuspended in Pfennig's hydrogen production medium by the method described in Materials and Methods. OD600 of hydrogen production culture was about 2.0.

At the beginning of hydrogen production stage, cells should be adapted for two days by the method described in Materials and Methods. After the adaptation, cells could convert 1 atm of carbon monoxide to hydrogen (Fig. 3a) and maximum CO conversion rate were reached at 5.3 mmol/L·hr(Fig. 3b). It has been reported that growth of all the microorganisms converting carbon monoxide to hydrogen is inhibited by carbon monoxide and begin after long lag period about 10 days. However, isolated strain converted 1 atm of carbon monoxide with only two-day adaption. In addition, conversion of carbon monoxide by the isolated strain was mass-transfer limited because the change of carbon conversion rate according to the partial pressure of carbon monoxide was almost linear. Maximum specific carbon monoxide conversion rate of this strain was estimated about 15 mmol/hr·g cells approximately and higher than that of Rhodospirillum rubrum which has highest maxium specific carbon monoxide conversion rate (11 mmol/hr·g cells) so far. Furthermore, doubling time was shorter than 1 hour  while  that  of  Rhodospirillum rubrum was about 5 hours.

Fig. 2 Chemoheterotrophic growth of isolated cell. Cell concentration was measured spectrophotometrically at 600 nm wavelength.

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