Novel Insights into Phosphorus Deprivation Boosted Lipid Synthesis in the Marine Alga Nannochloropsis oceanica without Compromising Biomass Production.

Nannochloropsis oceanica represents a preferred oleaginous alga for producing lipids. Here we found that phosphorus deprivation (PD) caused a severe decrease in protein and a considerable increase in lipids including triacylglycerol (TAG), yet it had little effect on the carbohydrate level and biomass production of N. oceanica. The combinatorial analysis by integrating physiological, biochemical, and transcriptomic data unraveled the molecular mechanisms underlying PD-induced lipid accumulation. Albeit attenuating the Calvin-Benson cycle, PD stimulated the C4-like pathway to maintain CO2 fixation for biomass production. PD attenuated nitrogen utilization and enhanced protein catabolism thus leading to protein decrease, from which the carbon was likely salvaged into the stimulated tricarboxylic acid cycle for supplying lipid synthesis with carbon precursors. The impairment of TAG catabolism by downregulating certain lipases rather than the stimulation of TAG assembly pathways contributed to PD-boosted TAG increase. These findings provide novel insights into PD-induced lipogenesis without compromising biomass production by N. oceanica.

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.

Development of a closed-loop process for fusel alcohol production and nutrient recycling from microalgae biomass.

Improving the economic feasibility is necessary for algae-based processes to achieve commercial scales for biofuels and bioproducts production. A closed-loop system for fusel alcohol production from microalgae biomass with integrated nutrient recycling was developed, which enables the reuse of nitrogen and phosphorus for downstream application and thus reduces the operational requirement for external major nutrients. Mixed fusel alcohols, primarily isobutanol and isopentanol were produced from Microchloropsis salina hydrolysates by an engineered E. coli co-culture. During the process, cellular nitrogen from microalgae biomass was converted into ammonium, whereas cellular phosphorus was liberated by an osmotic shock treatment. The formation of struvite from the liberated ammonium and phosphate, and the subsequent utilization of struvite to support M. salina cultivation was demonstrated. The closed loop system established here should help overcome one of the identified economic barriers to scale-up of microalgae production, and enhance the sustainability of microalgae-based chemical commodities production.

Enhanced production of carotenoids using a Thraustochytrid microalgal strain containing high levels of docosahexaenoic acid-rich oil.

Results to date suggest that microalgal Thraustochytrids family strains can be used to produce high-functional omega-3 rich oil (~ 30-70% of dry cell weight) and carotenoid-based antioxidant pigments simultaneously with value-added bioactive potential. In the present study, we describe the isolation and characterization of a new Thraustochytrid Schizochytrium sp. from the west coastal area of Korea. This newly isolated Thraustochytrid, identified as Schizochytrium sp. through 18S rRNA analysis and named SH104, simultaneously produces high levels of DHA and carotenoid-based antioxidant pigments. An improved Schizochytrium mutant, named SHG104, was obtained from the original host strain by γ-irradiation-induced mutagenesis. Under combined temperature-shift cultivation conditions employing white-light LEDs (light-emitting diodes), Schizochytrium sp. SHG104 yielded 10.8 g L-1 of biomass comprising 45.8% total lipids (32.1% DHA) and 4.6 mg L-1 of astaxanthin. In addition to DHA, the main fatty acids produced by Schizochytrium sp. SHG104 were palmitic acid and a trace of other long-chain fatty acids. The carotenoid profile of SH104 and SHG104 was ß-carotene, astaxanthin, canthaxanthin, pheonicoxanthin and echinenone, which analyzed by HPLC and LC/APCI-MS. Furthermore, genomic analysis of Schizochytrium and Aurantiochytrium microalgae confirmed that the presence of carotenogenesis pathway enzymes and genes including geranylgeranyl diphosphate, phytoene synthase, lycopene cyclase, and cytochrome P450 hydroxylase that necessary for the production of antioxidants via a complete biosynthetic KEGG synthesis pathway. This newly isolated Schizochytrium microalga potentially have wide application as a source of antioxidants for astaxanthin-containing pigments, commercial omega-3 lipids and feed additives, such as nutritional supplements for aquaculture.

Chattonella subsalsa (Raphidophyceae) growth and hemolytic activity in response to agriculturally-derived estuarine contaminants.

The potential for toxic contaminants and nutrient pollution to alter natural cycles of estuarine phytoplankton blooms is well known, yet few studies have examined how these combined stressors affect harmful algal species. Here, a robust testing protocol was developed to enable an ecotoxicological assessment of responses to commonly co-occurring estuarine contaminants by harmful algal bloom species. The population growth and toxicity (as cell density and hemolytic activity, respectively) of a cultured strain of the toxigenic raphidiophycean, Chattonella subsalsa, were assessed in two experiments (duration 10 days and 28 days) across a gradient of atrazine concentrations and N:P ratios simulating nutrient-rich versus nutrient-depleted regimes. The response of this large-celled, slowly growing alga to atrazine × nutrients depended on growth phase; atrazine was most inhibitory during early exponential population growth (day 10), whereas nutrient regime was a more important influence during later phases of growth (day 28). Without atrazine, toxicity toward fish was highest in low-P cultures. At atrazine levels >25 µg L-1, hemolytic activity was highest in low-N cultures, and increased with increasing atrazine concentration in all nutrient-limited cultures. Hemolytic activity varied inversely with atrazine concentration in N,P-replete conditions. Overall, atrazine inhibitory effects on population growth of this C. subsalsa strain depended on the growth phase and the nutrient regime; hemolytic activity was higher and further enhanced by atrazine in low N-P regimes; and atrazine inhibited hemolytic activity in nutrient-replete conditions. The data suggest that, depending on the growth phase and nutrient regime, atrazine can help promote toxic C. subsalsa blooms.

Metatranscriptome analysis reveals environmental and diel regulation of a Heterosigma akashiwo (raphidophyceae) bloom.

Despite numerous laboratory studies on physiologies of harmful algal bloom (HAB) species, physiologies of these algae during a natural bloom are understudied. Here, we investigated a bloom of the raphidophyte Heterosigma akashiwo in the East China Sea in 2014 using metabarcode (18S rDNA) and metatranscriptome sequencing. Based on 18S rDNA analyses, the phytoplankton community shifted from high diversity in the pre-bloom stage to H. akashiwo predominance during the bloom. A sharp decrease in ambient dissolved inorganic phosphate and strong up-regulation of phosphate and dissolved organic phosphorus (DOP) uptake genes, including the rarely documented (ppGpp)ase, in H. akashiwo from pre-bloom to bloom was indicative of rapid phosphorus uptake and efficient utilization of DOP that might be a driver of the H. akashiwo bloom. Furthermore, observed up-regulated expression of mixotrophy-related genes suggests potential contribution of mixotrophy to the bloom. Accelerating photosynthetic carbon fixation was also implied by the up-regulation of carbonic anhydrase genes during the bloom. Notably, we also observed a strong morning-to-afternoon shift in the expression of many genes. Our findings provide insights into metabolic processes likely important for H. akashiwo bloom formation, and suggest the need to consider timing of sampling in field studies on this alga.

Growth of mono- and mixed cultures of Nannochloropsis salina and Phaeodactylum tricornutum on struvite as a nutrient source.

The suitability of crude and purified struvite (MgNH4PO4), a major precipitate in wastewater streams, was investigated for renewable replacement of conventional nitrogen and phosphate resources for cultivation of microalgae. Bovine effluent wastewater stone, the source of crude struvite, was characterized for soluble N/P, trace metals, and biochemical components and compared to the purified mineral. Cultivation trials using struvite as a major nutrient source were conducted using two microalgae production strains, Nannochloropsis salina and Phaeodactylum tricornutum, in both lab and outdoor pilot-scale raceways in a variety of seasonal conditions. Both crude and purified struvite-based media were found to result in biomass productivities at least as high as established media formulations (maximum outdoor co-culture yield ∼20±4gAFDW/m(2)/day). Analysis of nutrient uptake by the alga suggest that struvite provides increased nutrient utilization efficiency, and that crude struvite satisfies the trace metals requirement and results in increased pigment productivity for both microalgae strains.

Integrated process of two stage cultivation of Nannochloropsis sp. for nutraceutically valuable eicosapentaenoic acid along with biodiesel.

The marine eustigmatophyte Nannochloropsis is one of the potential producers of eicosapentaenoic acid (EPA), a valued nutraceutical. Nannochloropsis sp. was cultivated under photoautotrophic condition utilizing CO2 in a two phase cultivation process in order to enhance the eicosapentaenoic acid (EPA) productivity. It was cultivated in a photobioreactor up to late log phase for cell growth (phase I). Then, the culture was harvested and confronted to relatively low temperature (10 °C) and low light (30 µmol photons m(-2) s(-1)) in both photobioreactor and Erlenmeyer flask (phase II), thus augmenting EPA% by 3.4 fold. Lower temperature with low light favored the synthesis of EPA although, biomass productivity, lipid content and lipid productivity were slightly decreased relative to phase I. The total lipids extracted from Nannochloropsis sp. fractionated into neutral lipids (NLs), glycolipids (GLs) and phospholipids (PLs) and a major proportion of EPA was found in phospholipids. Results suggested that low temperature and low light may ameliorate partitioning towards EPA in phospholipids.

Growth and alkaline phosphatase activity of Chattonella marina and Heterosigma akashiwo in response to phosphorus limitation.

The growth and alkaline phosphatase activity (APA) of two raphidophyceae species Chattonella marina and Heterosigma akashiwo were investigated in response to P-limitation and subsequent addition of dissolved inorganic phosphorus (DIP, NaH2PO4) and two dissolved organic phosphorus (DOP) compounds: guanosine 5-monophosphate (GMP) and triethyl phosphate (TEP). APA levels increased greatly after P-starvation as the decrease of the cellular phosphorus quotes (Qp). C. marina responded to P-limitation quickly and strongly, with 10-fold increase in APA within 24 hr after P-starvation. The larger difference between maximal and minimal QP values in C. marina indicated its high capacity in P storage. APA of H. akashiwo was maximally enlarged about 2.5 times at 48 hr of P-starvation. After the addition of nutrients, cell numbers of C. marina increased in all treatments including the P-free culture, demonstrating the higher endurance of C. marina to P-limitation. However, those of H. akashiwo increased only in DIP and GMP cultures. APA increased only after the addition of the monophosphate ester GMP. The results suggest that quick responses of C. marina to P-limitation, high capacity in P storage as well as endurance for P-depletion provide this species an ecological advantage in phytoplankton community competition under DIP-limited conditions.

Influence of the N:P supply ratio on biomass productivity and time-resolved changes in elemental and bulk biochemical composition of Nannochloropsis sp.

This work reports for the first time the detailed impacts of dual nitrogen (N) and phosphorus (P) stress on growth dynamics and biochemical composition in the Eustigmatophyte Nannochloropsis sp. P-stress concurrent with N-stress had subtle effects on culture bulk biochemical composition, but negatively influenced biomass productivity. However, the N:P supply ratio can be raised to at least 32:1 without compromising productivity (yielding a maximum lipid content of 52% of dry weight and volumetric lipid concentration of 233 mg L(-1)). The maximum biomass and lipid yields per unit of cell-P were 1.2 kg DW (gP)(-1) and 0.54 kg lipid (gP)(-1). The P concentration of many common media is thus in surplus for optimal Nannochloropsis sp. biomass and lipid production, offering potential for significant savings in P usage and improving the sustainability of algal cultivation.

A low-cost culture medium for the production of Nannochloropsis gaditana biomass optimized for aquaculture.

Nannochloropsis gaditana is a microalga with a high nutritional value and a protein and polyunsaturated fatty acid (PUFA) content that makes it interesting as a feed in aquaculture. To maximize its productivity and nutritional value in large-scale culture, a well-known commercial medium was optimized to the most favorable nutrient level using commercial fertilizers. Optimal growth conditions were obtained in the alternative fertilizer-based medium at a nitrogen concentration of 11.3 mM, a phosphorus concentration of 0.16 mM, and a micronutrient concentration of 30 µL L(-1). This alternative medium allowed to obtain a biomass concentration similar to that achieved when using the commercial formula but with a reduction in Cu, Fe, and Mo content of 71%, 89%, and 99%, respectively. A maximum biomass productivity of 0.51 g L(-1) d(-1) was obtained. The eicosapentaenoic acid and protein contents of the biomass were 2.84% and 44% of dry weight, respectively.

The central role of selenium in the biochemistry and ecology of the harmful pelagophyte, Aureococcus anophagefferens.

The trace element selenium (Se) is required for the biosynthesis of selenocysteine (Sec), the 21st amino acid in the genetic code, but its role in the ecology of harmful algal blooms (HABs) is unknown. Here, we examined the role of Se in the biology and ecology of the harmful pelagophyte, Aureococcus anophagefferens, through cell culture, genomic analyses, and ecosystem studies. This organism has the largest and the most diverse selenoproteome identified to date that consists of at least 59 selenoproteins, including known eukaryotic selenoproteins, selenoproteins previously only detected in bacteria, and novel selenoproteins. The A. anophagefferens selenoproteome was dominated by the thioredoxin fold proteins and oxidoreductase functions were assigned to the majority of detected selenoproteins. Insertion of Sec in these proteins was supported by a unique Sec insertion sequence. Se was required for the growth of A. anophagefferens as cultures grew maximally at nanomolar Se concentrations. In a coastal ecosystem, dissolved Se concentrations were elevated before and after A. anophagefferens blooms, but were reduced by >95% during the peak of blooms to 0.05 nM. Consistent with this pattern, enrichment of seawater with selenite before and after a bloom did not affect the growth of A. anophagefferens, but enrichment during the peak of the bloom significantly increased population growth rates. These findings demonstrate that Se inventories, which can be anthropogenically enriched, can support proliferation of HABs, such as A. anophagefferens through its synthesis of a large arsenal of Se-dependent oxidoreductases that fine-tune cellular redox homeostasis.

[Production of peroxide hydrogen in Chattonella ovata Hong Kong strain].

The growth characteristics of Chattonellea ovata Hong Kong strain (COHK) under different salinity and nutrient conditions and production of peroxide hydrogen (H2O2) in different growth phase were investigated in laboratory unialgal culture experiment. The results showed that high concentration of H2O2 was reached during the logarithmic phase from 4th to 8th days, corresponding to the maximum value of 2.91 x 10(-4) nmo x cell(-1) on the 6th day. When the N: P ratios were 16: 1, 32:1 and 64: 1, COHK possessed the relatively high growth rate and cell concentration, however, growth rate of COHK was lower with the N: P ratios of 4: 1 and 8: 1. The H2O2 concentration reached the highest (1.26 x 10(-4) nmol x cell(-1)) with the N: P ratio of 4: 1, which suggested that production of H2O2 was reversely related to the growth of COHK. The optimum salinities for COHK were 20 and 25. When the salinities were 10, 15 and 30, the growth rate of COHK was lower, showing lower and higher salinities inhibited the growth of COHK. Salinity also influenced the production of H2O2. The highest concentration of H2O2 among the experiments, 2. 2 x 10(-4) nmol x cell(-1), was observed at the salinity of 10, while there was no significant difference of H2O2 concentration from the salinity range of 15 to 30. The optimal Fe(3+) concentration for the growth of COHK was from 0.2 x 10(-8) to 1 x 10(-8) mol x L(-1), where growth rate were relatively higher. Growth rate of COHK was depressed in absence (0 mol x L(-1)) and high concentration of Fe3+ (5 x 10(-8) mol x L(-1)), with which H2O2 concentration was 0.97 x 10(-4) and 0.95 x 10(-4) nmol x cell(-1), respectively.