• ALGAE
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• v.28 no.1
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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.

• Journal of Korean Society of Water and Wastewater
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• v.27 no.6
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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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• v.30 no.10
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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.

• Microbiology and Biotechnology Letters
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• v.49 no.1
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• pp.101-110
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• 2021

Interactions between microalgae and heterotrophic bacteria are common in natural environments. This study investigated the effect of heterotrophic bacteria on the activity of the photosynthetic eukaryotic alga Chlorella sp. MF1907 when cocultured. A total of 31 heterotrophic bacterial isolates belonging to different genera were cocultured with MF1907. Interactions of the alga with Agromyces, Rhodococcus, Sphingomonas, Hyphomicrobium, Rhizobium, and Pseudomonas were positive, while those with Burkholderia, Paraburkholderia, Micrococcus, Arthrobacter, Mycobacterium, Streptomyces, Pedobacter, Mucilaginibacter, Fictibacillus, Tumebacillus, Sphingopyxis, and Erythrobacter were negative (p < 0.05). A turnover experiment demonstrating a switch from heterotrophic to autotrophic activity of MF1907 was performed using 16 isolates exhibiting apparent effects (positive, negative, or neutral). Compared with the results of the coculture experiment, eight isolates exhibited the same outcomes, while the others did not. Consistently, Pseudomonas and Agromyces showed a remarkable positive effect on MF1907 activity, and Burkholderia, Streptomyces, and Erythrobacter had a marked negative effect. Our results suggest that it may be possible to use the isolates for controlling populations of microalgae in natural and engineered environments.

• KSBB Journal
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• v.24 no.4
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• pp.377-382
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• 2009

The culture condition of growing Chlorella minutissima was optimized to produce biodiesel for fed-batch cultivation. First, under heterotrophic cultivation, the optimum level of glucose was determined to be 10 g/L for 20 days. After, three cultivation conditions were operated: autotrophic, heterotrophic, and mixotrophic growth. The lipid level and the maximum cell concentration from the fed-batch heterotrophic process were 32.0 (%, v/v) and 15.0 (g-dry wt./L) in 20 L flask, respectively. In addition, since the relatively constant specific lipid production rate was observed as 0.040 (% lipid/g-dry wt./day) at the latter period of cultivation time, the fed-batch process could maintain continuous lipid production. Fed-batch process is higher than those values from the batch process. The lipids from the fed-batch process contained over 38% of $C_{18}$, known as the suitable composition for the biodiesel application. For mixotrophic and heterotrophic growth under fed-batch condition, glucose was proved to be an appropriate carbon source for a large scale outdoor cultivation. For fed-batch cultivation, the feeding rate of seawater medium containing glucose was decided to be 0.5 L/day. The mixotrophic cultivation maintained maximum cell concentration of 24 (g-dry wt./L) and the lipid level of 43 (%, w/w). The lipid composition from this process was also proved to be suitable for the biodiesel production. The fatty acids from the mixotrophic growth contains 18% of $C_{17}$ and 49% of $C_{18}$, implying It also tells that C. minutissima is a suitable resource of biodiesel. Especially, the mixotrophic cultivation with fed-batch process might be useful for the large scale cultivation for the biodiesel production.

• Journal of Microbiology and Biotechnology
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• v.23 no.10
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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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• v.28 no.2
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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.

• Journal of Environmental Science International
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• v.12 no.8
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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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• v.32 no.1
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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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• v.27 no.11
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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.