알긴산염 마이크로캡슐 내부에 동결보존된 사상체 남세균의 특성 연구
Characterization of Filamentous Cyanobacteria Encapsulated in Alginate Microcapsules
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남세균은 수계나 토양 생태계에서 질소 고정 능력으로 인해 질소순환 과정에 중요한 역할을 하는 미생물로 다양한 산업분야에서 활용될 수 있는 유용 물질을 생산할 수 있다. 또한 일부 종은 수생태계에서 유해조류 번성을 일으켜 수자원의 이용을 제한한다. 일반적으로 생물소재은행은 관련 연구를 위해 남세균 배양주를 제공하는 역할을 한다. 하지만 국내 생물소재은행에서는 남세균의 체계적인 보존이 미비한실정이다. 본 연구에서는 국내에서 분리한 Trichormus variabilis (syn. Anabaena variabilis)를 대상으로 알긴산마이크로캡슐을 이용한 동결보존기술을 개발하였다. 동결보존제만 사용하는 일반 동결보존법과 세포를 마이크로캡슐에포집한 후 동결보존제를 혼합하는 두 가지 방법으로 실험을수행하였고 각각의 효율을 비교하였다. T. variabilis 동결보존 후 35일간 재배양하여 세포수를 비교한 결과 두 가지 동결보존법 모두 재생에 성공하였으며, 초기 농도 1.0 × 105 cells에서 마이크로캡슐 방법은 6.7 × 106 cells로, 일반 동결보존법은 1.1 × 107 cells로 증가되었다. 또한 배양 14일 후T. variabilis 세포의 부정형 형태를 발견하였다. 세포 소기관을 관찰하기 위해 투과전자현미경(TEM) 분석을 하였고lacunae와 lipid body의 크기가 줄어든 것을 확인하였다. 세포 형태와 관련된 mreB 유전자가 동결보존 전에 비해 동결보존 후 발현량이 54.7%로 감소된 것을 확인하였다. 본 연구결과를 통해 향후 사상성 남조류에 대하여 보존제와 마이크로캡슐을 이용한 동결보존이 가능할 것으로 기대된다.
Abstract
Cyanobacteria are microorganisms which have important roles in the nitrogen cycle due to their ability to fix nitrogen in water and soil ecosystems. They also produce valuable materials that may be used in various industries. However, some species of cyanobacteria may limit the use of water resources by causing harmful algal blooms in water ecosystems. Many culture collection depositories provide cyanobacterial strains for research, but their systematic preservation is not well-developed in Korea. In this study, we developed a method for the cryopreservation of the cyanobacteria Trichormus variabilis (syn. Anabaena variabilis), using alginate microcapsules. Two approaches were used for the experiments and their outputs were compared. One of the methods involved the cryopreservation of cells using only a cryoprotectant and the other used the cryoprotectant within microcapsules. After cryopreservation for 35 days, cells preserved with both methods were successfully regenerated from the initial 1.0 × 105 cells/ml to a final concentration of 6.7 × 106 cells/ml and 1.1 × 107 cells/ml. Irregular T. variabilis shapes were found after 14 days of regeneration. T. variabilis internal structures were observed by transmission electron microscopy (TEM), revealing that lipid droplets were reduced after cryopreservation. The expression of the mreB gene, known to be related to cell morphology, was downregulated (54.7%) after cryopreservation. Cryopreservation using cryoprotectant alone or with microcapsules is expected to be applicable to other filamentous cyanobacteria in the future.
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