Increased biomass and lipid production by continuous cultivation of Nannochloropsis salina transformant overexpressing a bHLH transcription factor.

Microalgae are promising feedstocks for sustainable and eco-friendly production of biomaterials, which can be improved by genetic engineering. It is also necessary to optimize the processes to produce biomaterials from engineered microalgae. We previously reported that genetic improvements of an industrial microalga Nannochloropsis salina by overexpressing a basic helix-loop-helix transcription factor (NsbHLH2). These transformants showed an improved growth and lipid production particularly during the early phase of culture under batch culture. However, they had faster uptake of nutrients, resulting in earlier starvation and reduced growth during the later stages. We attempted to optimize the growth and lipid production by growing one of the transformants in continuous culture with variable dilution rate and feed nitrogen concentration. Relative to wild-type, NsbHLH2 transformant consumed more nitrate at a high dilution rate (0.5 day -1 ), and had greater biomass production. Subsequently, nitrogen limitation at continuous cultivation led to an increased fatty acid methyl ester production by 83.6 mg l -1 day -1 . To elucidate genetic mechanisms, we identified the genes containing E-boxes, known as binding sites for bHLH transcription factors. Among these, we selected 18 genes involved in the growth and lipid metabolism, and revealed their positive contribution to the phenotypes via quantitative real-time polymerase chain reaction. These results provide proof-of-concept that NsbHLH2 can be used to produce biomass and lipids.

A mathematical model of intracellular behavior of microalgae for predicting growth and intracellular components syntheses under nutrient-replete and -deplete conditions.

Microalgae is a promising biomass source for renewable fuels and chemicals production. To describe microalgal behavior and improve their cultivation, various kinetic models have been proposed. However, previous works have focused on biomass formation and lipids production only, even though carbohydrates and proteins are also important products, not only for understanding the metabolic behavior of microalgae but also for enhancing the economic viability through value-added side products. In this study, a new mathematical model is proposed to explain core biological mechanisms of growth and macromolecules syntheses based on the central metabolism of carbon and nitrogen. In the model, microalgal growth is separated as hyperplasia and hypertrophy, to describe the cell growth more precisely under nutrient-replete and -deplete conditions. Sensitivity analysis performed using the model indicates that cell state (e.g., cell death rate) has a strong effect on the lipid production explaining the difficulty of reproducing a microalgae culture experiment.

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 lipid productivity by adopting multi-stage continuous cultivation strategy in Nannochloropsis gaditana.

In the present study, a novel process-based cultivation system was designed to improve lipid productivity of Nannochloropsis gaditana, an oleaginous microalga that has high potential for biofuel production. Specifically, four flat-panel photobioreactors were connected in series, and this system was subjected to continuous chemostat cultivation by feeding fresh medium to the first reactor at dilution rates of 0.028 and 0.056day-1, which were determined based on Monod kinetics. The results show that the serially connected photobioreactor system achieved 20.0% higher biomass productivity and 46.1% higher fatty acid methyl ester (FAME) productivity than a conventional single photobioreactor with equivalent dilution rate. These results suggest that a process-based approach using serially connected photobioreactors for microalgal cultivation can improve the productivity of lipids that can be used for biofuel production.

A simple method for decomposition of peracetic acid in a microalgal cultivation system.

A cost-efficient process devoid of several washing steps was developed, which is related to direct cultivation following the decomposition of the sterilizer. Peracetic acid (PAA) is known to be an efficient antimicrobial agent due to its high oxidizing potential. Sterilization by 2 mM PAA demands at least 1 h incubation time for an effective disinfection. Direct degradation of PAA was demonstrated by utilizing components in conventional algal medium. Consequently, ferric ion and pH buffer (HEPES) showed a synergetic effect for the decomposition of PAA within 6 h. On the contrary, NaNO3, one of the main components in algal media, inhibits the decomposition of PAA. The improved growth of Chlorella vulgaris and Synechocystis PCC6803 was observed in the prepared BG11 by decomposition of PAA. This process involving sterilization and decomposition of PAA should help cost-efficient management of photobioreactors in a large scale for the production of value-added products and biofuels from microalgal biomass.

Utilization of seawater for cost-effective cultivation and harvesting of Scenedesmus obliquus.

Microalgae hold great promise as a source of biofuels and biochemicals. The main obstacles to their industrial application are the high cultivation and downstream costs related to media and harvesting. In the work, we explored the multiple potentials of seawater to address key issues relating to the cultivation of Scenedesmus obliquus. Seawater can sufficiently replace some of the key elements in BG11 medium such as MgSO4, CaCl2, and NaCO3, and its use can significantly reduce the quantity of water required for the preparation of culture media. Among our results, the total chlorophyll content in cells grown in modified BG11 using 10 % (v/v) seawater was increased 1.47-fold without sacrificing biomass or lipid production. More than 70 % of the total algal biomass was auto-flocculated within one hour when cells were grown in seawater-supplemented media, which compares very favorably with a yield of only 3 % from cells grown in BG11.

Effect of monochromatic illumination on lipid accumulation of Nannochloropsis gaditana under continuous cultivation.

Although nitrogen starvation is frequently used to increase lipid contents in microalgae, it has a negative effect on cellular growth. Since light supply is essential for photosynthetic organisms, the effects of cultivation under monochromatic illumination on the growth and lipid contents of Nannochloropsis gaditana were assessed. Continuous cultivation under blue and red light conditions improved the productivity and physical properties for biodiesel from this microalga. FAME yield was twofold higher under red light than under normal white light (21.12% vs 11.35%), with no significant difference in growth rates. Blue and red light increased photosynthetic oxygen evolution, carbon fixation and nutrient uptake. In total, more significant physiological changes were observed under red than under blue light. These results show that red light illumination may be useful for enhancing lipid production by N. gaditana, with the increased photosynthetic reducing equivalents induced by red light which could be deposited as lipids and carbohydrates.

Optimization of tungsten dual poly-metal gates in memory devices with Ti/Wn/WSiN barrier metal.

Due to the demand of high-speed/high-density and low power application of memory devices, tungsten dual poly gate (W-DPG; W/barrier metals/n+ and p+ poly-Si) electrode could be a good solution in order to reduce gate sheet resistance (Rs). Process optimization is completed for a diffusion barrier metal in a W-DPG. A new noble WSiN layer is inserted between the Ti/WN barrier metal and the tungsten gate electrode to maintain large grain size of W deposited by physical vapor deposition. The annealed WSiN during post-processing changes into crystallized WSi(x) mixed with SiN, which can make vertical conductive path between top and bottom interface, contributing to low vertical contact resistance (Rc) and low gate Rs adequate for high speed requirement of memory device. The Ti/WN/WSiN barrier is found to have the same electrical performance, ring oscillator singal delay as complicated multi-layes barrier metal, Ti/WN/TiN/WSi(x)/WN reported earlier. Therefore, the gate stack can be optimized by introducing a simpler diffusion barrier metal.