Highly Efficient Genetic Transformation Methods for the Marine Oleaginous Diatom Fistulifera solaris.

The oleaginous diatom Fistulifera solaris is a promising producer of biofuel owing to the high content of the lipids. A genetic transformation technique by microparticle bombardment for this diatom was already established. However, the transformation efficiency was significantly lower than those of other diatoms. Devoting efforts to advance the genetic modifications of this diatom is crucial to unlock its full potential. In this study, we optimized the microparticle bombardment protocol, and newly established a multi-pulse electroporation protocol for this diatom. The nutrient-rich medium in the pre-culture stage played an essential role to increase the transformation efficiency of the bombardment method. On the other hand, use of the nutrient-rich medium in the electroporation experiments resulted in decreasing the efficiency because excess nutrient salts could hamper to establish the best conductivity condition. Adjustments on the number and voltage of the poring pulses were also critical to obtain the best balance between cell viability and efficient pore formation. Under the optimized conditions, the transformation efficiencies of microparticle bombardment and multi-pulse electroporation were 111 and 82 per 108 cells, respectively (37 and 27 times higher than the conventional bombardment method). With the aid of the optimized protocol, we successfully developed the transformant clone over-expressing the endogenous fat storage-inducing transmembrane protein (FIT)-like protein, which was previously found in the genome of the oleaginous diatom F. solaris and the oleaginous eustigmatophyte Nannochloropsis gaditana. This study provides powerful techniques to investigate and further enhance the metabolic functions of F. solaris by genetic engineering.

Assessment on the oil accumulation by knockdown of triacylglycerol lipase in the oleaginous diatom Fistulifera solaris.

Microalgae are promising producers of biofuel due to higher accumulation of triacylglycerol (TAG). However, further improvement of the lipid metabolism is critical for feasible application of microalgae in industrial production of biofuel. Suppression of lipid degradation pathways is a promising way to remarkably increase the lipid production in model diatoms. In this study, we established an antisense-based knockdown (KD) technique in the marine oleaginous diatom, Fistulifera solaris. This species has a capability to accumulate high content of lipids. Tgl1 KD showed positive impact on cell growth and lipid accumulation in conventional culture in f/2 medium, resulting in higher oil contents compared to wild type strain. However, these impacts of Tgl1 KD were slight when the cells were subjected to the two-stage growth system. The Tgl1 KD resulted in slight change of fatty acid composition; increasing in C14:0, C16:0 and C16:1, and decreasing in C20:5. This study indicates that, although Tgl1 played a certain role in lipid degradation in F. solaris, suppression of only a single type of TAG lipase was not significantly effective to improve the lipid production. Comprehensive understanding of the lipid catabolism in this microalga is essential to further improve the lipid production.

Algal biomass production by phosphorus recovery and recycling from wastewater using amorphous calcium silicate hydrates.

The phosphorous supply crisis is a major challenge for a sustainable society, and the algal industry is not unrelated to this crisis. Recycling phosphorus from sewage wastewater is a potential way to address this issue. We previously developed amorphous calcium silicate hydrates (aCSH) as excellent phosphorus recovery materials. In this study, we designed a phosphorus recovery process using aCSH in a pilot-scale facility connected to a sewage wastewater treatment plant, and demonstrated the production of microalgal biomass using phosphorous-containing aCSH (P_aCSH). As a result, high phosphorous recovery rates (>80%) were obtained throughout the year. The carbohydrate-rich microalga Pseudoneochloris sp. NKY372003 was cultivable with P_aCSH. The biomass and carbohydrate productivity of this microalga with P_aCSH was comparable to that with conventional media. Approximately 94% of the phosphorus in P_aCSH was recycled into the biomass. This study successfully demonstrated the recycling the phosphorus recovered from wastewater for microalgal cultivation by aCSH.

Selection and characterization of microalgae with potential for nutrient removal from municipal wastewater and simultaneous lipid production.

Wastewater treatment, along with the simultaneous production of valuable chemical compounds, including lipids by microalgae is a challenging but attractive study. Towards this goal, the candidate microalgae were selected from culture collections or isolated from wastewater in this study. The initial screening test using microalgae revealed that various eukaryotic as well as prokaryotic microalgae showed steady growth in municipal wastewater samples. Among them, Tetraselmis sp. NKG400013 and Parachlorella kessleri NKG021201 from culture collections, and Chloroidium saccharophilum NKH13 from the wastewater sample exhibited high biomass productivity. Furthermore, P. kessleri NKG021201 and C. saccharophilum NKH13 showed high lipid productivity (56 ± 1 mg/L/day for NKG021201, 35 ± 10 mg/L/day for NKH13). During this cultivation, 99% of nitrogen and 82% of phosphorous compounds were removed from the wastewater sample by the strain NKG021201. Analysis of fatty acid compositions of P. kessleri NKG021201 and C. saccharophilum NKH13 revealed that lipids derived from these microalgae were suitable for the application of biodiesel fuels, indicating that these microalgae were promising for wastewater treatment and lipid production.

Characterization of a novel marine unicellular alga, Pseudoneochloris sp. strain NKY372003 as a high carbohydrate producer.

Production of biofuels and fine chemicals from biomass-derived carbohydrates through biorefinery attracts much attention because it is recognized as an environmentally friendly process. Microalgae can serve as promising carbohydrate producers for biorefinery rather than woody and crop biomass due to high biomass productivity, high CO2 fixation, and no competition with food production. However, microalgae with high carbohydrate productivity have not been well investigated despite intensive studies of microalgal lipid production. In this study, the carbohydrate production of Pseudoneochloris sp. strain NKY372003 isolated as a high carbohydrate producer, was investigated. Cultivation conditions with various combinations of nutrient contents and photon flux density were examined to maximize the biomass and carbohydrate productivities. At the optimal condition, the biomass and carbohydrate production of this strain reached 8.11 ± 0.37 g/L and 5.5 ± 0.2 g/L, respectively. As far as we know, this is the highest carbohydrate production by microalgae among ever reported. Cell staining with Lugol's solution visualized intracellular starch granules. Because algal starch can be converted to biofuels and building blocks of fine chemicals, Pseudoneochloris sp. NKY372003 will be a promising candidate for production of fermentable carbohydrates towards biofuels and fine chemicals production.