Microalgae-derived biodiesel is perceived as the most promising candidate for next generation biofuel due to its sustainability and biodegradability. In this study, microalgal strain Chlamydmonas reinhardtii has been extensively evaluated and enhanced to be more suitable for biodiesel production. In order to upgrade C. reinhardtii, it was randomly mutagenized using a chemical mutagen, Ethyl Methane Sulfonate. As a result, a lipid-overproducing and fast-growing mutant #128 was screened. Its neutral lipid content was twice that of wild type throughout the growth period from exponential to N-depleted phase, when measured in terms of photoluminescence. Measured with flow cytometer, #128’s mean fluorescence was 866.51 compared to wild type’s 691.21. Confocal microscope images of BODIPY 505/515-stained cells clearly revealed that #128 contains larger and more abundant lipid bodies. Fatty acid content analyzed by gas chromatography also showed higher fatty acid content for #128 in exponential and stationary phase as well as after N-starvation. Mutant #128 contained higher quantities of C16:0, C16:1, C18:1n9c, C19:2n6t, and C18:3n6c fatty acids than the wild type. By N-starvation, fatty acid methyl ester (FAME) content of mutant #128 increased 2.3-fold while that of wild type increased 1.9-fold. As growth rate is another important factor for biodiesel commercialization, growth rate and lipid productivity were also analyzed. Not only did #128 accumulate more lipids, it also grew faster and accumulated more biomass. With the initial seeding dry cell weight of 0.10 g/L, #128 accumulated 0.93g/L of biomass compared to wild type’s 0.86 g/L. Lipid productivity of #128 reached 1.282 g L-1 d-1, whereas wild type reached 1.019 g L-1 d-1. From 2D gel electrophoresis and MALDI-TOF analysis of N-starved samples, it was found that beta-subunit of mitochondrial ATP Synthase and two-component response regulator PiIR may relate to #128’s superior characteristics.
While extensive studies are currently carried out on microalgae, evaluation of their lipid bodies is still in its infancy. Therefore, flow cytometry, confocal and fluorescence microscopies, and gas chromatography have been incorporated to thoroughly investigate and compare the intracellular lipid bodies of C. reinhardtii CC124 and its starchless mutant sta6-1. Using fluorescence-activated cell sorter (FACS), single cell sorting of microalgae was performed for the first time, yielding 79% survival rate for BODIPY-stained samples. Since flow cytometric results match gas chromatography results, flow cytometry was validated as another method of lipid evaluation. By comparing BODIPY 505/515 and Nile red, it was found that BODIPY 505/515 requires less concentration of organic solvent for staining, demonstrating itself as the better dye for flow cytometry of microalgae. With this study, it was determined that single cell sorting by FACS can isolate individual lipid-rich cells to seed new cultures.
미세조류 기반 바이오디젤은 지속가능성과 생분해성 덕분에 가장 각광받는 차세대 바이오연료다. 본 연구에서는 보다 효율적인 바이오디젤 생산을 위해 미세조류 Chlamydomonas reinhardtii를 분석하고 향상시키고자 하였다. 균주 개량을 위해서 Ethyl Methane Sulfonate를 이용하여 임의돌연변이를 유발하였고 지질 과잉생산 돌연변이 #128을 스크리닝하였다. Spectrophotofluorometer 측정 결과, #128은 모든 성장과정에 걸쳐 wild type 대비 2배의 중성지방을 축적하였으며 Flow cytometer로 측정한 #128의 평균 형광량은 866.51로 wild type의 691.21보다 높았다. Confocal 현미경 사진은 BODIPY 505/515 시약으로 염색한 #128의 lipid body가 훨씬 크고 많음을 보여주었고, gas chromatography (GC)로 측정한 지방산 함량 또한 #128이 모든 성장단계에서 높았다. 돌연변이 #128은 wild type 대비 더 높은 양의 C16:0, C16:1, C18:1n9c, C19:2n6t, C18:3n6c 지방산을 함유하였고 Nitrogen-starvation 조건 적용을 통해서 wild type은 fatty acid methyl ester (FAME) 양이 1.94배 증가한 반면 #128은 2.32배 증가함을 확인하였다. 바이오디젤의 상용화를 위해서는 성장속도 또한 중요한 요인이기에, 성장속도와 지질 생산성 또한 분석하였고, #128은 지질을 많이 생산하면서도 더 많은 양의 바이오매스를 축적하며 보다 빠른 성장을 나타내었다. 0.10 g/L의 초기 농도로 배양하여 wild type은 0.86 g/L, #128은 0.93 g/L의 바이오매스를 축적했고 wild type의 지질 생산성은 1.019 g L-1 d-1, #128은 1.282 g L-1 d-1였다.
현재 미세조류에 대해 많은 연구가 이루어지고 있지만, 그 lipid body에 대한 분석은 아직 초기 단계다. 그러므로 본 연구에서는 flow cytometer, confocal 및 형광 현미경, GC를 이용하여 클라미도모나스의 wild type CC124와 세포벽이 없는 sta6-1 돌연변이의 지질에 대한 철저한 분석을 수행하였다. 세포 자동해석 및 분리장치를 이용하여 미세조류의 single cell sorting이 최초로 이루어졌으며 BODIPY 505/515로 염색하여 분리된 샘플의 79%가 생존하였다. Flow cytometer 분석 결과 GC 결과와 유사하여, 미세조류에도 flow cytometer 분석이 가능함이 밝혀졌다. BODIPY 505/515와 Nile red 시약을 비교 분석한 결과, BODIPY 505/515가 더 적은 양의 유기용매를 필요로 하여 flow cytometry에 보다 적합한 시약임을 확인하였다. 본 연구를 통해 FACS를 이용한 고지질 세포 분리를 통한 새로운 종균 배양이 가능하다는 결론을 내릴 수 있었다.
