Desulfurization characteristics as well as hydrocarbon bioavailability of Gordonia nitida CYKS1 were investigated. CYKS1 could utilize long chain aliphatic hydrocarbons such as dodecane, hexadecane, and 2-, 6-, 10- and 14-tetramethylpentadecane for its growth. The activities of two desulfurization enzymes (DszA and DszC) and the enzyme for $FMNH_2$ regeneration (DszD) in cell extract were examined. The activities of DszA, DszC, and DszD from the cells incubated with DBT as the sole sulfur source were 8.0, 9.5 and 46.3 mU/mg, respectively. On the other hand, when the cells were cultured with $MgSO_4$, no activities of DszA and DszC were observed with a DszD activity of 27.18 mU/mg, suggesting that these desulfurization enzymes were induced by DBT. The activity of another desulfurization enzyme, DszB could not be measured for technical reasons. When LGO (light gas oil) was desulfurized by resting cells at various oil-to-water ratios (or oil fractions), its sulfur content only slightly decreased from 0.1 wt% to 0.09 wt% and no sulfate resulting from the desulfurization was detected in the aqueous phase, implying that CYKS1 uptake sulfur as much as for its cell growth only. For this reason, growing cells were used for the subsequent experiments.
An immobilized-cell air-lift reactor system was set up and its performance was investigated. A draft tube was placed inside the reactor and a patched type impeller was used for improving circulation of oil/water mixture. String-shaped nylon fiber bundles were used as the support for cell immobilization. The reactor performance was examined by repeated batch desulfurization of hydrotreated diesel oil. Sulfur content of diesel oil decreased from 500 ppm to 50 ppm at 10% of oil fraction and even at 50% of oil fraction. By GC-SCD chromatogram, it was identified that the remaining sulfur compounds were recalcitrant to CYKS1. Sucrose addition could improve the desulfurization efficiency at a high oil fraction. It was observed that 46% of n-alkanes was consumed when sucrose was not supplemented. However, only 33-37% of n-alkanes was utilized in the presence of sucrose. Continuous desulfurization was also conducted at different dilution rates and sulfur contents for up to 600 hours. The maximum desulfurization rate was 0.37 mg-sulfur/l/h, which was 1.5 times higher than that of batch desulfurization.
The whole desulfurization process including microbial desulfurization and phase separation was economically analyzed. The cost of desulfurization with an oil/water separator was 0.022-0.060 $/kg when the oil fraction was over 20% and the retention time was less than 3 hours. The direct fixed capital cost and equipment-dependent cost comprised the major parts in the capital investment and the annual operating cost, respectively. The cost of desulfurization with a phase separation with ethanol addition to facilitate phase separation was 0.028-0.068 $/kg. It was concluded that it was essential to improve the desulfurization activity of CYKS1 for the commercialization of biodesulfurization process.
As an effort to improve the desulfurization activity, a bacterial hemoglobin gene, vhb was transformed into a recombinant Streptomyces lividans TK24 with desulfurization genes, to enhance DBT oxidation rate. The specific DBT oxidation rate of the recombinant S. lividans TK24 with desulfurization genes and vhb gene was 0.109 μM/h/g-cell, which is about two-fold higher than that of the recombinant of S. lividans TK24 with only desulfurization genes. An RT-PCR analysis was carried out for the cells cultured in an R2YE medium containing $SO_4^{2-}$ and an SMM medium containing DBT as the sole sulfur source. It was observed that mRNAs of the desulfurization genes were expressed even in the case of the cells incubated in the R2YE medium, suggesting at least that $SO_4^{2-}$ had not repressed the transcription of desulfurization genes and that there was a high chance that $SO_4^{2-}$ might have inhibited some of the desulfurization enzymes. The expression of the vhb gene was also confirmed.
본 연구에서 사용한 Gordonia nitida CYKS1의 탈황특성 및 탄화수소의 생물학적 이용도 (hydrocarbon bioavailability)를 조사하였다. CYKS1은 dodecane, hexadecane, 2-, 6-, 10-, 14-tetramethylpentadecane과 같은 긴 사슬구조의 탄화수소를 성장을 위하여 탄소원으로 이용할 수 있었다. 두 개의 탈황효소 (DszA와 DszC)와 $FMNH_2$ 재생효소 (DszD)의 활성을 세포추출물 (cell extract)를 이용하여 측정하였다. DBT를 황원으로 배양된 세포에서 얻은 DszA, DszC, DszD의 활성은 각각 8.0, 9.5, 46.3 mU/mg이었다. 반면, $MgSO_4$ 를 사용하여 배양한 세포에서 얻은 DszA와 DszC의 활성은 나타나지 않았으며, DszD의 활성은 27.18 mU/mg이었다. 이것은 탈황효소의 활성이 DBT에 의해 유도 (induction)된다는 것을 의미한다. 또 다른 탈황효소인 DszB의 활성은 기술적인 문제로 측정할 수 없었다. LGO (light gas oil)를 다양한 유상비에서 휴식세포 (resting cell)를 이용하여 탈황하였을 때, 디젤유의 황함량을 0.1 wt%에서 0.09wt%로 줄일 수 있었으며, 탈황과정에서 생길 수 있는 황산염 ($SO_4^{2-}$) 을 수상에서 발견할 수는 없었다. 이는 CYKS1이 단지 세포 성장에 필요한 만큼의 황을 섭취한다는 것을 의미하며, 이러한 이유로, 이후의 실험에서는 성장세포 (growing cell)를 이용하였다.
디젤유 탈황을 위하여 세포고정화 공기부양반응기 (immobilized-cell air-lift reactor)를 개발하고 성능을 평가하였다. 반응기 내부에 draft tube를 설치하고 유류 및 배양액의 원할한 순환을 위하여 patched type impeller를 사용하였다. 또한 나일론 섬유 묶음을 세포 고정화를 위한 담체로 사용하였다. HDS 처리를 한 디젤유를 반복회분식 탈황 (repeated batch desulfurization) 공정을 거쳐 반응기의 성능을 시험하였다. 디젤유의 황함량을 유상비가 10%일 때, 500ppm에서 50ppm으로 감소시켰으며, 유상비 50%일 때에도 200ppm까지 감소시킬 수 있었다. GC-SCD chromatogram을 볼 때, 남아있는 황화합물들은 CYKS1이 제거하기 힘든 것들임을 알 수 있었다. 높은 유상비에서는 자당을 첨가함으로써 탈황효율을 증가시킬 수 있었다. 또한, 자당을 공급하지 않았을 경우에는 46%의 n-alkanes이 탄소원으로 이용되었으나, 자당을 공급한 경우에는 33-37%만의 n-alkanes이 소모되었다. 연속탈황공정 (continuous desulfurization)을 다양한 희석속도 (dilution rate)와 황함량에서 600시간 동안 수행하였다. 최대 탈황속도는 0.37 mg-sulfur/l/h 이었으며, 회분식 탈황 속도에 비해 1.5배 향상되었다.
미생물을 이용한 탈황공정과 유류, 배양액, 세포의 분리공정을 포함하는 전체 탈황공정의 경제성 평가를 수행하였다. 유/수상 분리장치를 이용한 경우의 탈황비용은 유상비 20%이상, 체류시간 3시간 이내의 조건에서 0.022-0.060 $/kg이었다. Direct fixed capital과 equipment-dependent cost가 각각 capital invest와 annual operating cost의 대부분을 차지하였다. 유수상 분리를 원할하게 하기 에탄올을 첨가하는 경우의 탈황비용은 0.028-0.068 $/kg였다. 따라서, 미생물을 이용한 탈황공정을 상업하기 위해서는 CYKS1의 탈황성능을 향상시키는 것이 필수적인 것으로 판단되었다.
탈황성능을 향상시키는 한 방법으로, 탈황유전자를 도입시킨 재조합 Streptomyces lividans TK24에 DBT 산화속도를 증가시키기 위하여 bacterial hemoglobin gene (vhb)을 추가로 도입시켰다. 탈황유전자와 헤모글로빈 유전자가 도입된 재조합 S. lividans TK24의 DBT 비산화속도는 0.109 μM/h/g-cell였으며, 탈황유전자만을 도입시틴 재조합 S. lividans TK24의 비산화속도보다 약 두 배 향상되었다. 황원으로 $SO_4^{2-}$ 이 있는 R2YE 배지와 DBT를 포함한 SMM 배지에서 배양한 세포를 이용하여 RT-PCR 분석을 수행하였다. R2YE 배제에서 배양한 세포의 경우에도 탈황유전자의 mRNA가 발현되었으며, 이는 적어도 $SO_4^{2-}$ 이 탈황유전자의 전사를 억압하지는 않으며, 탈황효소의 활성을 저해할 가능성이 높다는 것을 의미한다. 또한 헤모글로빈 유전자의 발현도 확인하였다.
