Transcriptional insights into Chlorella sp. ABC-001: a comparative study of carbon fixation and lipid synthesis under different CO2 conditions.

BACKGROUND: Microalgae's low tolerance to high CO2 concentrations presents a significant challenge for its industrial application, especially when considering the utilization of industrial exhaust gas streams with high CO2 content-an economically and environmentally attractive option. Therefore, the objectives of this study were to investigate the metabolic changes in carbon fixation and lipid accumulation of microalgae under ambient air and high CO2 conditions, deepen our understanding of the molecular mechanisms driving these processes, and identify potential target genes for metabolic engineering in microalgae. To accomplish these goals, we conducted a transcriptomic analysis of the high CO2-tolerant strain, Chlorella sp. ABC-001, under two different carbon dioxide levels (ambient air and 10% CO2) and at various growth phases. RESULTS: Cells cultivated with 10% CO2 exhibited significantly better growth and lipid accumulation rates, achieving up to 2.5-fold higher cell density and twice the lipid content by day 7. To understand the relationship between CO2 concentrations and phenotypes, transcriptomic analysis was conducted across different CO2 conditions and growth phases. According to the analysis of differentially expressed genes and gene ontology, Chlorella sp. ABC-001 exhibited the development of chloroplast organelles during the early exponential phase under high CO2 conditions, resulting in improved CO2 fixation and enhanced photosynthesis. Cobalamin-independent methionine synthase expression was also significantly elevated during the early growth stage, likely contributing to the methionine supply required for various metabolic activities and active proliferation. Conversely, the cells showed sustained repression of carbonic anhydrase and ferredoxin hydrogenase, involved in the carbon concentrating mechanism, throughout the cultivation period under high CO2 conditions. This study also delved into the transcriptomic profiles in the Calvin cycle, nitrogen reductase, and lipid synthesis. Particularly, Chlorella sp. ABC-001 showed high expression levels of genes involved in lipid synthesis, such as glycerol-3-phosphate dehydrogenase and phospholipid-diacylglycerol acyltransferase. These findings suggest potential targets for metabolic engineering aimed at enhancing lipid production in microalgae. CONCLUSIONS: We expect that our findings will help understand the carbon concentrating mechanism, photosynthesis, nitrogen assimilation, and lipid accumulation metabolisms of green algae according to CO2 concentrations. This study also provides insights into systems metabolic engineering of microalgae for improved performance in the future.

Light Stress after Heterotrophic Cultivation Enhances Lutein and Biofuel Production from a Novel Algal Strain Scenedesmus obliquus ABC-009.

Scenedesmus obliquus ABC-009 is a microalgal strain that accumulates large amounts of lutein, particularly when subjected to growth-limiting conditions. Here, the performance of this strain was evaluated for the simultaneous production of lutein and biofuels under three different modes of cultivation - photoautotrophic mode using BG-11 medium with air or 2% CO2 and heterotrophic mode using YM medium. While it was found that the highest fatty acid methyl ester (FAME) level and lutein content per biomass (%) were achieved in BG-11 medium with CO2 and air, respectively, heterotrophic cultivation resulted in much higher biomass productivity. While the cell concentrations of the cultures grown under BG-11 and CO2 were largely similar to those grown in YM medium, the disparity in the biomass yield was largely attributed to the larger cell volume in heterotrophically cultivated cells. Post-cultivation light treatment was found to further enhance the biomass productivity in all three cases and lutein content in heterotrophic conditions. Consequently, the maximum biomass (757.14 ± 20.20 mg/l/d), FAME (92.78 ± 0.08 mg/l/d), and lutein (1.006 ± 0.23 mg/l/d) productivities were obtained under heterotrophic cultivation. Next, large-scale lutein production using microalgae was demonstrated using a 1-ton open raceway pond cultivation system and a low-cost fertilizer (Eco-Sol). The overall biomass yields were similar in both media, while slightly higher lutein content was obtained using the fertilizer owing to the higher nitrogen content.

Strategic implementation of phosphorus repletion strategy in continuous two-stage cultivation of Chlorella sp. HS2: Evaluation for biofuel applications.

Lipid production in microalgae under nitrogen (N) starved condition can be enhanced by excess phosphorus (P) supply in the second stage of two-stage cultivation strategy. However, implementing two-stage cultivation is difficult in large-scale cultivation system as it requires high energy of transferring large algal biomass from first stage to second stage. To address this problem, we have optimized a continuous two-stage (CTS) cultivation strategy using Chlorella sp. HS2, where nitrogen in the growth environment is depleted naturally via consumption. To enhance both biomass and lipid productivity this strategy explored supplementation of additional P from 50% to 2500% of the initial concentration at the start of N-limited second stage of growth. The results of the optimization study in photobioreactor (PBR) showed that supplementing 500% of initial P and 100% of initial other nutrients (O) (N0-P500-O100) on 5th day showed the maximum biomass productivity of 774.4 mg L-1 d-1. It was observed that Chlorella sp. HS2 grown in PBR yielded higher biomass (3.8 times), lipid (6.1 times) and carbohydrate (5.5 times) productivity in comparison to the open raceway ponds (ORP) study, under optimum nutrient and carbon supply condition. The maximum lipid (289.6 mg L-1 d-1) and carbohydrate (219.2 mg L-1 d-1) productivities were obtained in TPBR-3, which were 1.9 and 1.3 times higher than that of TPBR-2 (+ve control) and 9.6 and 3.7 times higher than that of TPBR-1 (-ve control), respectively. Fatty acid mainly composed of C16/C18 (84.5%-85.7%), which makes the microalgal oil suitable for biofuel production. This study concluded that feeding excess amount of P is an effective and scalable strategy to improve the biomass and lipid productivity of CTS cultivation.

Plant anti-aging: Delayed flower and leaf senescence in Erinus alpinus treated with cell-free Chlorella cultivation medium.

Plant tissues naturally senesce over time. Attempts to improve plant robustness and increase longevity have involved genetic modification, application of synthetic chemicals, and use of beneficial microbes. Recently, culture supernatant from a microalga Chlorella fusca was found to prime innate immunity against Pseudomonas syringae in Arabidopsis thaliana. However, the capacity of Chlorella culture supernatants to prevent or delay aging in higher plants has not been elucidated. In this study, roots of the ornamental flowering plant Erinus alpinus L. were drenched with cell-free supernatants from three Chlorella species. Flower and leaf senescence in E. alpinus was significantly reduced and delayed with all three Chlorella supernatants. Investigations of the mode of action underlying delayed senescence showed that the Chlorella supernatants did not act as a chemical trigger to elicit plant immunity or as a growth-promoting fertilizer in E. alpinus. The mechanisms underlying the anti-aging effects remain undetermined, and several possible hypotheses are discussed. Several Chlorella species are industrially cultivated, and disposal of cell-free supernatant can be economically and environmentally challenging. This study provides a novel method for extending plant lifespan through use of Chlorella supernatant and discusses the potential of using industrial waste supernatants in agriculture and horticulture to reduce reliance on chemical pesticides and genetic modification.

Author Correction: Application of biosurfactant from Bacillus subtilis C9 for controlling cladoceran grazers in algal cultivation systems.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Optimization of heterotrophic cultivation of Chlorella sp. HS2 using screening, statistical assessment, and validation.

The heterotrophic cultivation of microalgae has a number of notable advantages, which include allowing high culture density levels as well as enabling the production of biomass in consistent and predictable quantities. In this study, the full potential of Chlorella sp. HS2 is explored through optimization of the parameters for its heterotrophic cultivation. First, carbon and nitrogen sources were screened in PhotobioBox. Initial screening using the Plackett-Burman design (PBD) was then adopted and the concentrations of the major nutrients (glucose, sodium nitrate, and dipotassium phosphate) were optimized via response surface methodology (RSM) with a central composite design (CCD). Upon validation of the model via flask-scale cultivation, the optimized BG11 medium was found to result in a three-fold improvement in biomass amounts, from 5.85 to 18.13 g/L, in comparison to a non-optimized BG11 medium containing 72 g/L glucose. Scaling up the cultivation to a 5-L fermenter resulted in a greatly improved biomass concentration of 35.3 g/L owing to more efficient oxygenation of the culture. In addition, phosphorus feeding fermentation was employed in an effort to address early depletion of phosphate, and a maximum biomass concentration of 42.95 g/L was achieved, with biomass productivity of 5.37 g/L/D.

Biological Carbon Recovery from Sugar Refinery Washing Water into Microalgal DHA: Medium Optimization and Stress Induction.

Sugar refinery washing water (SRWW) contains abundant levels of carbon sources and lower levels of contaminants than other types of wastewater, which makes it ideal for heterotrophic cultivation of microalgae. Here, carbon sources in SRWW were utilized for conversion into the form of value-added docosahexaenoic acid (DHA) using Aurantiochytrium sp. KRS101. Since SRWW is not a defined medium, serial optimizations were performed to maximize the biomass, lipid, and DHA yields by adjusting the nutrient (carbon, nitrogen, and phosphorus) concentrations as well as the application of salt stress. Optimum growth performance was achieved with 30% dilution of SRWW containing a total organic carbon of 95,488 mg L-1. Increasing the nutrient level in the medium by supplementation of 9 g L-1 KH2PO4 and 20 g L-1 yeast extract further improved the biomass yield by an additional 14%, albeit at the expense of a decrease in the lipid content. Maximum biomass, lipid, and DHA yields (22.9, 6.33, and 2.03 g L-1, respectively) were achieved when 35 g L-1 sea salt was applied on a stationary phase for osmotic stress. These results demonstrate the potential of carbon-rich sugar refinery washing water for DHA production using Aurantiochytrium sp. KRS101 and proper cultivation strategy.

Efficient microalgae removal from aqueous medium through auto-flocculation: investigating growth-dependent role of organic matter.

This study investigated the growth-dependent role of algal organic matters (AOMs) to achieve high removal efficiency (R.E) of microalgae. The results showed that the microalgae cells produced 96 ± 2% of total AOMs as loose bound AOMSS (LB-AOMs) and 4 ± 1% as cell-bound (CB-AOMs) in exponential phase. In stationary phase, LB-AOMs and CB-AOMs were 46 ± 0.7percentage and 54 ± 0.2 percentage, respectively. The R.Es in exponential and stationary phase were 83 ± 2.6% and 66 ± 1.2%, respectively. It is found that the difference of biomass concentration (between exponential and stationary phase) had no significant impact on the R.E (P > 0.01). Further investigations revealed that LB-AOMs inhibit flocculation in exponential and CB-AOMs in stationary phase; however, CB-AOMs showed stronger inhibition than the LB-AOMs (P < 0.01). The provision of calcium (17 ± 0.9 mg/L) to the culture reduced the AOMs inhibition and improved the R.E from 66 ± 1.2% (in control) to 90 ± 4.2%. An increase in R.E was attributed to the interaction of calcium with AOMs and subsequently acting as a flocculant. The findings of this study can be valuable to improve the performance of auto-flocculation technology, which is mainly limited by the presence of AOMs. Graphical Abstract.

Author Correction: Statistical optimization of light intensity and CO2 concentration for lipid production derived from attached cultivation of green microalga Ettlia sp.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Isolation and Characterization of Novel Chlorella Species with Cold Resistance and High Lipid Accumulation for Biodiesel Production.

Chlorella spp. are green algae that are found across wide-ranging habitats from deserts to arctic regions, with various strains having adapted to survive under diverse environmental conditions. In this study, two novel Chlorella strains (ABC-002, ABC-008) were isolated from a freshwater lake in South Korea during the winter season and examined for possible use in the biofuel production process. The comparison of ABC-002 and ABC-008 strains with Chlorella vulgaris UTEX265 under two different temperatures (10°C, 25°C) revealed their cold-tolerant phenotypes as well as high biomass yields. The maximum quantum yields of UTEX25, ABC- 002, and ABC-008 at 10°C were 0.5594, 0.6747, and 0.7150, respectively, providing evidence of the relatively higher cold-resistance capabilities of these two strains. Furthermore, both the biomass yields and lipid content of the two novel strains were found to be higher than those of UTEX265; the overall lipid productivities of ABC-002 and ABC-008 were 1.7 ~ 2.8 fold and 1.6 ~ 4.2 fold higher compared to that of UTEX265, respectively. Thus, the high biomass and lipid productivity over a wide range of temperatures indicate that C. vulgaris ABC-002 and ABC-008 are promising candidates for applications in biofuel productions via outdoor biomass cultivation.

Increased biomass and lipid production of Ettlia sp. YC001 by optimized C and N sources in heterotrophic culture.

The culture conditions and media composition for the heterotrophic culture of an axenic strain of Ettlia sp. YC001 were firstly optimized using the Plackett-Burman design (PBD) and response surface methodology (RSM). The strain successfully showed higher productivity in the basal media without any light illumination at 32.2 to 33.3 °C. The PBD results showed that the most effective components for biomass productivity of Ettlia sp. were fructose and yeast extract for sources of C and N, respectively. The RSM results showed an optimal level of 72.2 g/L for fructose and 21.5 g/L for yeast extract, resulting in 46.1 g/L biomass with a lipid content of 13.8% over a course of 9 days. Using a 5 L scaled-up fermentation system for 6 days, the production of biomass and lipids was 7.21 g/L/day and 1.18 g/L/day, respectively. Consequently, heterotrophic cultivation of Ettlia sp. YC001 provided much higher production of biomass and lipids than those of autotrophic cultivation. As further research, the use of substitute substrates instead of fructose and yeast extract should be developed to reduce production costs.

Exploration of two-stage cultivation strategies using nitrogen starvation to maximize the lipid productivity in Chlorella sp. HS2.

In this work, the two-stage cultivation of Chlorella sp. HS2 for enhancing the lipid productivity was optimized by adjusting the duration of nitrogen-replete (N+) and -deplete (N-) stages within a 9 day period using urea as nitrogen source. The highest lipid content of 36.7% and productivity of 216.9 mg L-1 d-1 were obtained under five days of N+ followed by four days of N- conditions. Replenishing phosphorus and other nutrients (N-P+O+) at the beginning of the nutrient-starvation resulted in 1.55 and 1.68-folds improvement in lipid productivities compared to the single stage and zero nutrient controls (N-P-O-), respectively. The estimated biodiesel properties based on the fatty acid profiles met all criteria of international standards. The findings of this study indicate that properly adjusting the period of nitrogen availability as well as the presence of other nutrients is highly important in order to maximize the biofuel productivity in two-stage microalgal cultivation.

Statistical optimization of light intensity and CO2 concentration for lipid production derived from attached cultivation of green microalga Ettlia sp.

Attached cultivation systems have been receiving extensive attention as a breakthrough in microalgae cultivation technology. However, there is a lack of studies that emphasize precise optimization of important parameters in attached cultivation of microalgae. In this study, the effects of two major environmental parameters in photoautotrophic cultivation, light intensity and CO2 concentration, on the biomass and lipid surface productivity of Ettlia sp. YC001 were optimized by employing Response Surface Methodology (RSM) and validated experimentally. The optimum initial conditions for attached cultivation were use of seed from the late exponential phase (LE) and an inoculum surface density of 2.5 g/m2. By optimization, maximum biomass surface productivity of 28.0 ± 1.5 g/m2/day was achieved at 730 µE/m2/s with 8% CO2. The maximum lipid surface productivity was 4.2 ± 0.3 g/m2/day at 500 µE/m2/s with 7% CO2. Change of the fatty acid composition with respect to changes in environment parameters led to improvement of biodiesel quality at higher light intensity and higher CO2 concentration. Attached cultivation of Ettlia sp. YC001 has successfully produced biomass and lipids at a high production rate with relatively low light energy demand and high CO2 utilization.

MAPK/ERK and JNK pathways regulate lipid synthesis and cell growth of Chlamydomonas reinhardtii under osmotic stress, respectively.

Microalgae have great potential for the production of biofuels due to the ability of the organism to accumulate large quantities of storage lipids under stress conditions. Mitogen activated protein kinase (MAPK) signaling cascades are widely recognized for their role in stress response signal transduction in eukaryotes. To assess the correlation between MAPK activation and lipid productivity, Chlamydomonas reinhardtii was studied under various concentrations of NaCl. The results demonstrated that C. reinhardtii exhibits elevated levels of extracellular-signal regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) activities after undergoing osmotic stress, as well as an increase in cellular lipid content. To establish a more direct causal link between both kinases and lipid productivity, C. reinhardtii was subjected to biochemically induced regulation of ERK and JNK pathways. Activating the MEK-ERK pathway via C6 ceramide treatment increased ERK activation and lipid production simultaneously, while PD98059 mediated inhibition of the pathway yielded opposite results. Interestingly, suppression of the JNK pathway with SP600125 resulted in a substantial decrease in cell viability under osmotic stress. These results suggest that ERK and JNK MAP kinases have important roles in microalgal lipid accumulation and cell growth under osmotic stress, respectively.

Advanced multigene expression system for Nannochloropsis salina using 2A self-cleaving peptides.

Even though there has been much interest in genetic engineering of microalgae, its progress has been slow due to the difficulty and limitation of available techniques. Currently, genetic modification in most microalgal strains is confined to single gene transformation. Here, a multigene expression system for the oleaginous model strain Nannochloropsis salina was developed with glycine-serine-glycine spacer linked 2A self-cleaving peptides (2A) for the first time. An efficiency test of the four most widely used 2As revealed that two different types of 2As T2A and E2A have the best performance in N. salina with a maximum cleavage rate of nearly 45%. The system was able to express the linked sequence of the selection marker shble and the fluorescence protein sfCherry with intact functions. Because 2A enabled multigene expression in the single cassette form, the use of 2A also reduced the vector size, which along with the stronger promoter resulted in a 9-fold increase in the transformation efficiency. Furthermore, confirmative screening accuracy of more than 90% was observed. Hence, the 2A applied vector system is expected to be beneficial in microalgal research field because it enables multigene expression as well as offering improved transformation and screening efficiency.

Application of biosurfactant from Bacillus subtilis C9 for controlling cladoceran grazers in algal cultivation systems.

Open algal cultivation platforms often suffer crop losses to herbivorous grazers that have potential to devastate biomass production within a few days. While a number of studies suggest synthetic chemicals as control agents for voracious algal grazers, environmental and safety concerns associated with the use of these chemicals encourage the exploration of alternative biological control agents. We hereby propose the application of a biosurfactant produced by Bacillus subtilis C9 (referred to as C9-biosurfactant) for controlling cladoceran grazers commonly found in algal cultivation systems. The results indicated that C9-biosurfactant completely eradicated Daphnia pulex and Moina macrocopa within 24 hours when concentrations were equal to or exceeded 6 mg/L. Moreover, supplying C9-biosurfactant into the cultures of selected algal species with and without cladoceran grazers indicated no adverse effect of C9-biosurfactant on the growth and lipid productivity of algal crops, while cladocerans were selectively controlled by C9-biosurfactant even under the presence of their prey. These results thus indicate that C9-biosurfactant could be an effective biocontrol agent for cladoceran grazers at industrial algal cultivation.

A simulation study on the effects of neuronal ensemble properties on decoding algorithms for intracortical brain-machine interfaces.

BACKGROUND: Intracortical brain-machine interfaces (BMIs) harness movement information by sensing neuronal activities using chronic microelectrode implants to restore lost functions to patients with paralysis. However, neuronal signals often vary over time, even within a day, forcing one to rebuild a BMI every time they operate it. The term "rebuild" means overall procedures for operating a BMI, such as decoder selection, decoder training, and decoder testing. It gives rise to a practical issue of what decoder should be built for a given neuronal ensemble. This study aims to address it by exploring how decoders' performance varies with the neuronal properties. To extensively explore a range of neuronal properties, we conduct a simulation study. METHODS: Focusing on movement direction, we examine several basic neuronal properties, including the signal-to-noise ratio of neurons, the proportion of well-tuned neurons, the uniformity of their preferred directions (PDs), and the non-stationarity of PDs. We investigate the performance of three popular BMI decoders: Kalman filter, optimal linear estimator, and population vector algorithm. RESULTS: Our simulation results showed that decoding performance of all the decoders was affected more by the proportion of well-tuned neurons that their uniformity. CONCLUSIONS: Our study suggests a simulated scenario of how to choose a decoder for intracortical BMIs in various neuronal conditions.

Wavelength shift strategy to enhance lipid productivity of Nannochloropsis gaditana.

BACKGROUND: Microalgae, being a phototroph, grow in the presence of light, and utilizing photons in narrow and specific range of wavelengths. There have been numerous attempts to take advantage of this trait of wavelength-dependent growth for the purpose of increasing biomass productivity. One potential option involves wavelength conversion of sunlight. In the present study, three fluorescent dyes with blue, red, and green emission spectra were employed with the aim of improving sunlight utilization efficiency and thus enhancing biomass and lipid productivity of Nannochloropsis gaditana. RESULTS: When DPA and R101 were used to enrich blue and red spectra, biomass productivity of Nannochloropsis gaditana was increased by 35.1 and 40.3%, respectively. The maximum quantum yield values were higher than 0.6 at the early stage of growth for the cultures grown under DPA- and R101-modified solar radiation. Chlorophyll a content was also 57.0 and 32.3% higher than the control at the early growth stage under DPA- and R101-modified solar radiation, respectively. This stimulation of photosynthetic activity at the early growth stage correlated well with rapid growth under DPA- and R101-modified light during the first 4 days of cultivation. Lipid productivity consequently increased by 26.9 (DPA) and 39.4% (R101) after 10 days of cultivation. An immediate effect on lipid induction was observed in cultures under modified light, which exhibited 19.1% improvement in lipid content at the cost of some degree of impaired growth. CONCLUSION: Fluorescent dyes with the capability of enriching wavelengths of light favored by the algal photosystem could indeed be an effective means of promoting growth of Nannochloropsis gaditana. This strategy would be particularly powerful for mass cultivation where sunlight is the only economically viable option for illumination.

Enhancement of biomass and lipid productivity by overexpression of a bZIP transcription factor in Nannochloropsis salina.

Microalgae are considered as excellent platforms for biomaterial production that can replace conventional fossil fuel-based fuels and chemicals. Genetic engineering of microalgae is prerequisite to maximize production of materials and to reduce costs for the production. Transcription factors (TFs) are emerging as key regulators of metabolic pathways to enhance production of molecules for biofuels and other materials. TFs with the basic leucine zipper (bZIP) domain have been known as stress regulators and are associated with lipid metabolism in plants. We overexpressed a bZIP TF, NsbZIP1, in Nannochloropsis salina, and found that transformants showed enhanced growth with concomitant increase in lipid contents. The improved phenotypes were also notable under stress conditions including N limitation and high salt. To understand the mechanism underlying improved phenotypes, we analyzed expression patterns of predicted target genes involved in lipid metabolism via quantitative RT-PCR, confirming increases transcript levels. NsbZIP1 appeared to be one of type C bZIPs in plants that has been known to regulate lipid metabolism under stress. Taken together, we demonstrated that NsbZIP1 could improve both growth and lipid production, and TF engineering can serve as an excellent genetic engineering tool for production of biofuels and biomaterials in microalgae.

Complementation of a mutation in CpSRP43 causing partial truncation of light-harvesting chlorophyll antenna in Chlorella vulgaris.

Photosynthesis of microalgae enables conversion of light energy into chemical energy to produce biomass and biomaterials. However, the efficiency of this process must be enhanced, and truncation of light-harvesting complex (LHC) has been suggested to improve photosynthetic efficiency. We reported an EMS-induced mutant (E5) showing partially reduced LHC in Chlorella vulgaris. We determined the mutation by sequencing the whole genome of WT and E5. Augustus gene prediction was used for determining CDS, and non-synonymous changes in E5 were screened. Among these, we found a point mutation (T to A) in a gene homologous to chloroplast signal recognition particle 43 kDa (CpSRP43). The point mutation changed the 102nd valine to glutamic acid (V102E) located in the first chromodomain. Phylogenetic analyses of CpSRP43 revealed that this amino acid was valine or isoleucine in microalgae and plants, suggesting important functions. Transformation of E5 with WT CpSRP43 showed varying degrees of complementation, which was demonstrated by partial recovery of the LHCII proteins to the WT level, and partially restored photosynthetic pigments, photosynthetic ETR, NPQ, and growth, indicating that the V102E mutation was responsible for the reduced LHC in E5.