• ALGAE
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• 제28권1호
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• pp.101-109
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• 2013

Some Chlorella species grow heterotrophically with organic substrate in dark condition. However, heterotrophic Chlorella species are limited and their optimum culture conditions are not fully known. In this study, three heterotrophic Chlorella species, two strains (C4-3 and C4-4) of C. vulgaris and one Chlorella sp. (C4-8) were examined on optimum culture conditions such as carbon source, temperature, and concentrations of nitrogen and phosphorus in Jaworski's medium (JM). And the growth and fatty acid composition of Chlorella were analyzed. For three heterotrophic Chlorella species, glucose (1-2%) as a carbon source only increased the growth and the range of optimum culture temperature was $26-28^{\circ}C$. Doubled concentrations of the nitrogen or phosphorus in JM medium also improved the growth of Chlorella. Chlorella cultured heterotrophically showed significantly higher growth rate and bigger cell size than those autotrophically did. C. vulgaris (C4-3) cultured heterotrophically showed the highest biomass in dry weight ($0.8g\;L^{-1}$) among three species. With respect to fatty acid composition, the contents of C16:0 and n-3 highly unsaturated fatty acid (HUFA) were significantly higher in autotrophic Chlorella than in heterotrophic one and those of total lipid were not different between different concentrations of nitrogen and phosphorus in JM medium. Among three Chlorella species in this study, C. vulgaris (C4-3) appeared to be the most ideal heterotrophic Chlorella species for industrial application since it had a high biomass and lipid content.

• 상하수도학회지
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• 제27권6호
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• pp.703-709
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• 2013

Among many microalgae cultivation types, heterotrophic culture with low cost carbon sources and energy saving culture method is crucial. A result of estimating the effects of pH on wastewater treatment using heterotrophic growing microalgae Chlorella sorokiniana shows that there was no difference in microalgae growth amount and nitrogen, phosphorus removal rate by wide range of pH(5 ~ 9). From pH 5 to 9, total nitrogen, phosphorous and glucose removal rates were 10.5 mg-N/L/d, 2 mg-P/L/d, 800 ~ 1000 mg/L respectively. This study reveals that C. sorokiniana cannot metabolite glycerol heterotrophically, however, glucose and acetate were proper carbon sources for growth and T-N, T-P and TOC removal. This research highlights the potential of heterotrophic microalgal growth with wastewater treatment plant with wide range of pH and carbon sources.

• Journal of Microbiology and Biotechnology
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• 제30권10호
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• pp.1597-1606
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• 2020

Transcription factor engineering to regulate multiple genes has shown promise in the field of microalgae genetic engineering. Here, we report the first use of transcription factor engineering in Chlorella sp. HS2, thought to have potential for producing biofuels and bioproducts. We identified seven endogenous bZIP transcription factors in Chlorella sp. HS2 and named them HSbZIP1 through HSbZIP7. We overexpressed HSbZIP1, a C-type bZIP transcription factor, in Chlorella sp. HS2 with the goal of enhancing lipid production. Phenotype screening under heterotrophic conditions showed that all transformants exhibited increased fatty acid production. In particular, HSbZIP1 37 and 58 showed fatty acid methyl ester (FAME) yields of 859 and 1,052 mg/l, respectively, at day 10 of growth under heterotrophic conditions, and these yields were 74% and 113% higher, respectively, than that of WT. To elucidate the mechanism underlying the improved phenotypes, we identified candidate HSbZIP1-regulated genes via transcription factor binding site analysis. We then selected three genes involved in fatty acid synthesis and investigated mRNA expression levels of the genes by qRT-PCR. The result revealed that the possible HSbZIP1-regulated genes involved in fatty acid synthesis were upregulated in the HSbZIP1 transformants. Taken together, our results demonstrate that HSbZIP1 can be utilized to improve lipid production in Chlorella sp. HS2 under heterotrophic conditions.

• 한국미생물·생명공학회지
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• 제49권1호
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• pp.101-110
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• 2021

다양한 환경에서 미세조류와 종속영양세균(heterotrophic bacteria) 사이의 상호작용은 일반적이다. 본 연구에서는 미세조류 Chlorella sp. MF1907와 서로 다른 속(genus)에 속하는 31종의 종속영양세균을 공배양(co-culture)하여 MF1907의 광합성 활성에 미치는 종속영양세균의 영향을 규명하였다. 6종의 종속영양세균(Agromyces, Rhodococcus, Sphingomonas, Hyphomicrobium, Rhizobium, Pseudomonas)은 MF1907의 광합성 활성을 증가시켰으며(p < 0.05), 12종의 종속영양세균(Burkholderia, Paraburkholderia, Micrococcus, Arthrobacter, Mycobacterium, Streptomyces, Pedobacter, Mucilaginibacter, Fictibacillus, Tumebacillus, Sphingopyxis, Erythrobacter)은 MF1907의 광합성 활성을 저해하였다(p < 0.05). 종속영양세균 중 MF1907의 광합성 활성에 유의미한 효과(positive, negative, neutral)를 나타낸 16종을 선택하여 MF1907의 종속영양에서 독립영양으로의 활성 전환에 미치는 이들의 영향을 평가하였다. 8종의 종속영양세균은 공배양 결과와 동일하게 MF1907의 종속영양에서 독립영양으로의 활성 전환에 영향을 미쳤다. 하지만 나머지 8종은 공배양 결과와 MF1907의 종속영양에서 독립영양으로의 활성 전환에 미치는 결과가 반대를 나타내었다. 공배양과 활성 전환 실험 모두에서 일관되게 Pseudomonas와 Agromyces는 MF1907의 광합성 활성을 강하게 증가시켰으며(p < 0.05), Burkholderia, Streptomyces, Erythrobacter는 MF1907의 광합성 활성을 강하게 저해하였다(p < 0.05). 본 연구 결과는 다양한 종속영양세균과 미세조류 사이의 상호작용 이해를 도모하고 종속영양세균을 활용하여 자연 환경과 공정 시스템에서 미세조류의 바이오매스를 조절할 수 있음을 시사한다.

• KSBB Journal
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• 제24권4호
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• pp.377-382
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• 2009

본 연구는 다양한 조건에서의 실험을 위한 C. minutissima의 500 mL flask의 종 배양을 통해 고농도의 균주 획득 후, 가장 효율적인 배양 방식을 보인 fed-batch를 통한 autotrophic, heterotrophic, mixotrophic 조건하에서 바이오디젤용 지질 생산의 최적 배양 조건을 찾아야했다. 먼저, fed-batch의 Heterotrophic 배양 시 유기 탄소 원으로 glucose의 가장 효율적인 안정한 초기농도가 10 g/L임을 확인했다. 이 후, 20 L 배양기를 이용한 batch, fed-batch의 heterotrophic 배양에서 fed-batch배양의 경우 지질 생성양이 1.62배 정도 더 높았으며, 최대 균체량 역시 4 (g-dry wt./L)정도 높으며, 또한 디젤용 지질로 적합한 $C_{18}$의 조성 역시 49%이상 차지하여, 옥외 대량 배양 시 유기탄소 원으로 적절한 glucose의 이용을 통한 효율적인 배양방법임을 알 수 있다. 이 결과를 바탕으로, glucose를 포함한 배지를 0.5 L/day로 공급하여 유가 식 배양을 각 배양 조건에 따라 비교 실시했다. 그 결과 mixotrophic의 경우 다른 배양 조건에 비해 각각 24 (g-dry wt./L)의 최대 군체 농도와 함께 43 (%, w/w)의 높은 지질 생성량을 보였다. 또한 생성되는 지질의 조성이 균체 당 바이오 디젤용 양질의 지질 생성이 가능하였다. 또한 fed-batch 배양 이후 0.047 (% lipid/g-dry wt./day)의 비 생산 속도를 유지하며, 일정 균체 농도를 유지하는 경우 지속적인 지질 생성이 가능함을 보여주었다. 이러한 세 가지 배양 조건하에서 지질 추출을 통한 지방산 조성을 살펴본 결과 mixotrophic 배양 하에 디젤용으로 잘 알려진 $C_{17}$, $C_{18}$의 지방산 조성은 각각 18%와 49%의 생성을 보여주었다. 이 결과들을 바탕으로 이 균주를 이용해 옥외 배양이나 배양 조에서 대량 배양이 가능함을 확인했으며, 특히, 옥외배양을 위한 혼합영양 배양조건하에 배양 공정의 scale-up에 대한 추가적인 연구를 통해 C. minutissima 미세조류로부터 바이오 디젤의 보다 경제적 생산이 가능하도록 하고자 한다.

• Journal of Microbiology and Biotechnology
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• 제23권10호
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• pp.1460-1471
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• 2013

Microalgal biofuel production from wastewater has economic and environmental advantages. This article investigates the lipid production from high chemical oxygen demand (COD) bioethanol wastewater without dilution or additional nutrients, using a newly isolated heterotrophic microalga, Chlorella vulgaris LAM-Q. To enhance lipid accumulation, the combined effects of important operational parameters were studied via response surface methodology. The optimal conditions were found to be temperature of $22.8^{\circ}C$, initial pH of 6.7, and inoculum density of $1.2{\times}10^8cells/ml$. Under these conditions, the lipid productivity reached 195.96 mg/l/d, which was markedly higher than previously reported values in similar systems. According to the fatty acid composition, the obtained lipids were suitable feedstock for biodiesel production. Meanwhile, 61.40% of COD, 51.24% of total nitrogen, and 58.76% of total phosphorus were removed from the bioethanol wastewater during microalgal growth. In addition, 19.17% of the energy contained in the wastewater was transferred to the microalgal biomass in the fermentation process. These findings suggest that C. vulgaris LAM-Q can efficiently produce lipids from high-concentration bioethanol wastewater, and simultaneously performs wastewater treatment.

• ALGAE
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• 제28권2호
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• pp.131-147
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• 2013

Major progress has been made in the past decade towards understanding of the biosynthesis of red carotenoid astaxanthin and its roles in stress response while exploiting microalgae-based astaxanthin as a potent antioxidant for human health and as a coloring agent for aquaculture applications. In this review, astaxanthin-producing green microalgae are briefly summarized with Haematococcus pluvialis and Chlorella zofingiensis recognized to be the most popular astaxanthin-producers. Two distinct pathways for astaxanthin synthesis along with associated cellular, physiological, and biochemical changes are elucidated using H. pluvialis and C. zofingiensis as the model systems. Interactions between astaxanthin biosynthesis and photosynthesis, fatty acid biosynthesis and enzymatic defense systems are described in the context of multiple lines of defense mechanisms working in concert against photooxidative stress. Major pros and cons of mass cultivation of H. pluvialis and C. zofingiensis in phototrophic, heterotrophic, and mixotrophic culture modes are analyzed. Recent progress in genetic engineering of plants and microalgae for astaxanthin production is presented. Future advancement in microalgal astaxanthin research will depend largely on genome sequencing of H. pluvialis and C. zofingiensis and genetic toolbox development. Continuous effort along the heterotrophic-phototrophic culture mode could lead to major expansion of the microalgal astaxanthin industry.

• 한국환경과학회지
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• 제12권8호
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• pp.847-852
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• 2003

It was examined carefully that the bacterial die-off between Chlorella vulgaris and E. coli. W3110 was tested through adding TOC (total organic carbon) with the lab-scaled continuous river water flow system (CRWFS). Artificial synthetic wastewater was applied at two levels of organic carbon concentration; 1,335 mg/l in treatment type 1 and 267 mg/l in type 2. In both types, the population densities of Chlorella vulgaris were similar in a maximum 8.25 ${\times}$ 10$\^$6/ cells/ml (type 1) and 6.925 ${\times}$ 10$\^$6/ cells/ml (type 2). The maximum densities of E. coli. W3110 were 2.0 ${\times}$ 10$\^$8/ colony forming unit (CFU)/ml in type 1 and 3.9 ${\times}$ 10$\^$8/ CFU/ml in type 2. The densities increased for 11 days in type 1 and 4 days in type 2, then decreased rapidly till the 35th day, then slightly increased again. This trend was prominent in type 2. It implied that a wider range of nutrients was required in the growth of heterotrophic bacteria in type 2 than in type 1. We could not expect successful bacterial die-off if the wastewater retention time was not furnished sufficiently.

• ALGAE
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• 제32권1호
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• pp.67-74
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• 2017

The heterotrophic dinoflagellate Oxyrrhis marina is known to produce high levels of docosahexaenoic acid (DHA) when fed on diverse algal prey. However, large-scale culturing of algal prey species is not easy and requires a large amount of budget, and thus more easily cultivable and low-cost prey is required. Dried yeast was selected as a strong candidate for an alternative prey in our preliminary tests. Thus, we explored the fatty acid composition and DHA production of O. marina fed on dried yeast and compared these results to those of O. marina fed on two algal prey species: the phototrophic dinoflagellate Amphidinium carterae and chlorophyte Chlorella sp. powder. O. marina fed on dried yeast, which does not contain DHA, produced the same high level of DHA as those fed on DHA-containing A. carterae. This indicates that O. marina is likely to produce DHA by itself regardless of prey items. Furthermore, the DHA content (and portion of total fatty acid methyl esters) of O. marina satiated with dried yeast, 52.40 pg per cell(and 25.9%), was considerably greater than that of O. marina fed on A. carterae (26.91 pg per cell; 15.7%) or powder of Chlorella sp. powder (21.24 pg per cell; 16.7%). The cost of dried yeast (approximately 10 US dollars for 1 kg dried yeast) was much lower than that of obtaining the algal prey (approximately 160 US dollars for 1 kg A. carterae). Therefore, compared to conventional algal prey, dried yeast is a more easily obtainable and lower-cost prey for use in the production of DHA by O. marina.

• Journal of Microbiology and Biotechnology
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• 제27권11호
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• pp.2010-2018
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• 2017

Mixotrophic microalgal growth gives a great premise for wastewater treatment based on photoautotrophic nutrient utilization and heterotrophic organic removal while producing renewable biomass. There remains a need for a control strategy to enrich them in a photobioreactor. This study performed a series of batch experiments using a mixotroph, Chlorella sorokiniana, to characterize optimal guidelines of mixotrophic growth based on a statistical design of the experiment. Using a central composite design, this study evaluated how temperature and light irradiance are associated with $CO_2$ capture and organic carbon respiration through biomass production and ammonia removal kinetics. By conducting regressions on the experimental data, response surfaces were created to suggest proper ranges of temperature and light irradiance that mixotrophs can beneficially use as two types of energy sources. The results identified that efficient mixotrophic metabolism of Chlorella sorokiniana for organics and inorganics occurs at the temperature of $30-40^{\circ}C$ and diurnal light condition of $150-200{\mu}mol\;E{\cdot}m^{-2}{\cdot}s^{-1}$. The optimal specific growth rate and ammonia removal rate were recorded as 0.51/d and 0.56/h on average, respectively, and the confirmation test verified that the organic removal rate was $105mg\;COD{\cdot}l^{-1}{\cdot}d^{-1}$. These results support the development of a viable option for sustainable treatment and effluent quality management of problematic livestock wastewater.