Tetraselmis Subcordiformis Research Articles

Application of an in situ CO2\u2013bicarbonate system under nitrogen depletion to improve photosynthetic biomass and starch production and regulate amylose accumulation in a marine green microalga Tetraselmis subcordiformis

BackgroundMicroalgal starch is regarded as a promising alternative to crop-based starch for biorefinery such as the production of biofuels and bio-based chemicals. The single or separate use of inorganic carbon source, e.g., CO2 and NaHCO3, caused aberrant pH, which restricts the biomass and starch production. The present study applied an in situ CO2–NaHCO3 system to regulate photosynthetic biomass and starch production along with starch quality in a marine green microalga Tetraselmis subcordiformis under nitrogen-depletion (−N) and nitrogen-limitation (±N) conditions.ResultsThe CO2 (2%)–NaHCO3 (1 g L−1) system stabilized the pH at 7.7 in the −N cultivation, under which the optimal biomass and starch accumulation were achieved. The biomass and starch productivity under −N were improved by 2.1-fold and 1.7-fold, respectively, with 1 g L−1 NaHCO3 addition compared with the one without NaHCO3 addition. NaHCO3 addition alleviated the high-dCO2 inhibition caused by the single CO2 aeration, and provided sufficient effective carbon source HCO3− for the maintenance of adequate photosynthetic efficiency and increase in photoprotection to facilitate the biomass and starch production. The amylose content was also increased by 44% under this CO2–bicarbonate system compared to the single use of CO2. The highest starch productivity of 0.73 g L−1 day−1 under −N cultivation and highest starch concentration of 4.14 g L−1 under ±N cultivation were both achieved with the addition of 1 g L−1 NaHCO3. These levels were comparable to or exceeded the current achievements reported in studies. The addition of 5 g L−1 NaHCO3 under ±N cultivation led to a production of high-amylose starch (59.3% of total starch), which could be used as a source of functional food.ConclusionsThe in situ CO2–NaHCO3 system significantly improved the biomass and starch production in T. subcordiformis. It could also regulate the starch quality with varied relative amylose content under different cultivation modes for diverse downstream applications that could promote the economic feasibility of microalgal starch-based biofuel production. Adoption of this system in T. subcordiformis would facilitate the CO2 mitigation couple with its starch-based biorefinery.

Phosphorus-rich microorganism-enabled synthesis of cobalt phosphide/carbon composite for bisphenol A degradation through activation of peroxymonosulfate

Phosphorus (P) recovery is of significant importance due to its limited reserves and non-regenerative nature. In this study, we attempted to recover and utilize P from microorganism to obtain metal phosphide/carbon composites. By P-rich cultivation of a model yeast (Candida utilis), a total 4.3 wt% P could be accumulated in microorganism. A hydrothermal treatment coupled with pyrolysis approach enabled the formation of cobalt phosphide/carbon (Co2P/C) composites using P-rich microorganism and Co2+ as precursors. Thermogravimetric-mass spectrometry (TG-MS) analysis confirmed the generation of reducing gases (CO, CH4, PH3, etc.) in pyrolysis process, which played important roles for the conversion of phosphate (in microorganism) to phosphide (in Co2P). When adopted as catalyst for peroxymonosulfate (PMS) activation, the developed Co2P/C composite performed well for bisphenol A (BPA) degradation with a 97.8% removal efficiency in 30 min. The recyclability of Co2P/C was also evident by five successive recycle runs. Moreover, intermediates in BPA degradation process were identified by gas chromatography-mass spectrometry (GC–MS) and liquid chromatography-mass spectrometry (LC-MS), which allowed us to propose a BPA degradation pathway. The following preliminary ecotoxicity test using Tetraselmis subcordiformis as ecological indicator confirmed that BPA degradation solution was much less toxic than the original solution. The strategy proposed in this study, which is the utilization of integrated elements (P, C, etc.) in microorganism to develop efficient catalyst, may provide a new solution for the recovery and value-added conversion of biomass/biowaste.

Flocculation activity of carp protamine in microalgal cells

Protamine, a polymer cationic peptide, originates from fish milt, an aquatic bioresource produced in massive quantities as a byproduct of fish processing. Protamine has been used as a heparin antagonist, hemostatic agent in the medical field and as an antimicrobial agent in the food industry. However, few studies have examined its flocculation activity. To investigate the applications of protamine extracted from carp (Cyprinus carpio Linnaeus) as a flocculating agent in microalgal cells, protamine extracts were prepared by digestion with sulfuric acid and purified by ion-exchange chromatography. The results showed that purified protamine was rich in lysine (39.24%) but low in arginine (1.92%). Carp protamine showed good flocculation activity towards Tetraselmis subcordiformis, with the flocculation rate reaching 85.00% at a protamine concentration of 20 mg/L. However, lower flocculation rates were observed for Dunaliella salina (23.56%) and Chlorella vulgaris (9.65%) at the same protamine concentration. After flocculation, precipitated T. subcordiformis cells were viable and could be recultured in fresh medium, showing similar growth curves as the original culture. Additionally, residual cells in the upper liquid were recultured and flocculated by protamine again, with a flocculation rate reaching 84.20% at a protamine concentration of 20 mg/L. The flocculating effect may be related in part to the electrostatic affinity of protamine to the negative surface charges of microalgal cells. The flocculation activity of carp protamine showed potential for use in harvesting of microalgal cells in the aquaculture industry. Furthermore, our results may have applications in the development of new approaches for promoting the high-value utilization of fish milt.

Phosphorus Enhances Photosynthetic Storage Starch Production in a Green Microalga (Chlorophyta) Tetraselmis subcordiformis in Nitrogen Starvation Conditions.

Microalgae are potential starch producers as alternatives to agricultural crops. This study disclosed the effects and mechanism of phosphorus availability exerted on storage starch production in a starch-producing microalga Tetraselmis subcordiformis in nitrogen starvation conditions. Excessive phosphorus supply facilitated starch production, which differed from the conventional cognition that phosphorus would inhibit transitory starch biosynthesis in plants. Phosphorus enhanced energy utilization efficiency for biomass and storage starch production. ADP-glucose pyrophosphorylase (AGPase), conventionally known to be critical for starch biosynthesis, was negatively correlated to storage starch biosynthesis. Excessive phosphorus supply maintained large cell volumes, enhanced activities of starch phosphorylases (SPs) along with branching enzymes and isoamylases, and increased phosphoenolpyruvate and trehalose-6-phosphate levels to alleviate the inhibition of high phosphate availability to AGPase, all of which improved starch production. This work highlighted the importance of phosphorus in the production of microalgal starch and provided further evidence for the SP-based storage starch biosynthesis pathway.

Characterization of starch phosphorylase from the marine green microalga (Chlorophyta) Tetraselmis subcordiformis reveals its potential role in starch biosynthesis

In a marine green starch-producing microalga Tetraselmis subcordiformis, the role of starch phosphorylase (SP) in the starch biosynthesis was disclosed by characterizing the enzyme properties and activity variations during the starch accumulation process. TsSP4, a SP isoform accounting for the major SP activity in T. subcordiformis, was unique to be active in a monomer form with a molecular weight of approximately 110kDa. It resembled one of the chloroplast-located SPs (PhoA) in Chlamydomonas reinhardtii with a similarity of 63.3% in sequence, though it possessed the typical L78/80 domain found in the plastidial SPs (Pho1) of higher plants that was absent in PhoA. TsSP4 exhibited moderate sensitivity to ADP-Glc inhibition and had a high activity for longer-chain linear maltooligosacchride (MOS) and amylopectin against highly branched glycogen as the substrates. TsSP4 had 2-fold higher affinity for Glc-1-P in the synthetic direction than for Pi in the phosphorolytic direction, and the catalytic constant kcat for Glc-1-P was 2-fold of that for Pi. Collectively, TsSP4 preferred synthetic rather than phosphorolytic direction. TsSP4 could elongate MOSs even initially with Pi alone in the absence of Glc-1-P, which further supported its synthetic role in the starch biosynthesis. TsSP4 displayed increased activities in the developing and mature stage of starch biosynthesis under nitrogen-starvation conditions, indicating its possible contribution to the amylopectin amplification.

Effect of Light/Dark Regimens on Hydrogen Production by Tetraselmis subcordiformis Coupled with an Alkaline Fuel Cell System.

To improve the photoproduction of hydrogen (H2) by a green algae-based system, the effect of light/dark regimens on H2 photoproduction regulated by carbonyl cyanide m-chlorophenylhydrazone (CCCP) was investigated. A fuel cell was integrated into a photobioreactor to allow online monitoring of the H2 evolution rate and decrease potential H2 feedback inhibition by consuming the generated H2 in situ. During the first 15h of H2 evolution, the system was subjected to dark treatment after initial light illumination (L/D=6/9h, 9/6h, and 12/3h). After the dark period, all systems were again exposed to light illumination until H2 evolution stopped. Two peaks were observed in the H2 evolution rate under all three light/dark regimens. Additionally, a high H2 yield of 126±10mLL-1 was achieved using a light/dark regimen of L 9h/D 6h/L until H2 production ceased, which was 1.6 times higher than that obtained under continuous illumination. H2 production was accompanied by some physiological and morphological changes in the cells. The results indicated that light/dark regimens improved the duration and yield of H2 photoproduction by the CCCP-regulated process of Tetraselmis subcordiformis.

Transient expression of the enhanced green fluorescence protein (egfp) gene in Tetraselmis subcordiformis (Chlorodendrales, Chlorophyta) with three exogenous promoters

:A stable transformation system for Tetraselmis subcordiformis had been established, but a low transformation rate limits its application to this alga. As a promoter is a key factor for transgenic research, a suitable promoter could increase the transformation rate. To improve a transformation system for T. subcordiformis, three commonly used promoters for algal transformation were selected. The CMV, CaMV35S and SV40 promoters were transferred to T. subcordiformis by glass-bead agitation and particle bombardment, with the enhanced green fluorescence protein (egfp) gene as the reporter gene. The transformation rate was calculated according to the GFP fluorescence in transformed algae. Results showed that the CaMV 35S promoter transformation efficiency was higher than that of CMV and SV40 promoters. This report will contribute to efforts to establish a high-efficiency transformation system and expands the fundamental research and applications of this alga.

Construction of a growth model in the green alga Tetraselmis subcordiformis using a response surface approach

The green alga Tetraselmis subcordiformis has been widely used as a quality live food for aquaculture species, and also has been studied as a model organism for the photo-biological production of hydrogen. We attempted to quantify the relationship between T. subcordiformis specific growth rate (SGR) and three important environmental factors (temperature, salinity, and pH) using the central composite design and response surface method under laboratory conditions. The results showed that the linear effects of temperature and salinity were significant (P 0.05); the interactive effect of temperature and pH was significant (P 0.05); all of the quadratic effects of the three factors were significant (P<0.05). A model equation for specific growth rate with the three factors was established, with the unadjusted and predictive R2 as high as 0.990 and 0.921, respectively, suggesting that the model was a very good fit and that it could be used to predict SGR. Through optimizing the reliable model, an optimal 3-factor combination of 25 °C/35 of salinity/pH 7.9 was obtained, at which the maximum specific growth rate (0.65) was recorded, with a desirability value of 93.8%. These experimental results could serve as guidelines for increasing T. subcordiformis production efficiency.

Enhanced green fluorescent protein (egfp) gene expression in Tetraselmis subcordiformis chloroplast with endogenous regulators.

On the basis of fundamental genetic transformation technologies, the goal of this study was to optimize Tetraselmis subcordiformis chloroplast transformation through the use of endogenous regulators. The genes rrn16S, rbcL, psbA, and psbC are commonly highly expressed in chloroplasts, and the regulators of these genes are often used in chloroplast transformation. For lack of a known chloroplast genome sequence, the genome-walking method was used here to obtain full sequences of T. subcordiformis endogenous regulators. The resulting regulators, including three promoters, two terminators, and a ribosome combination sequence, were inserted into the previously constructed plasmid pPSC-R, with the egfp gene included as a reporter gene, and five chloroplast expression vectors prepared. These vectors were successfully transformed into T. subcordiformis by particle bombardment and the efficiency of each vector tested by assessing EGFP fluorescence via microscopy. The results showed that these vectors exhibited higher efficiency than the former vector pPSC-G carrying exogenous regulators, and the vector pRFA with Prrn, psbA-5'RE, and TpsbA showed the highest efficiency. This research provides a set of effective endogenous regulators for T. subcordiformis and will facilitate future fundamental studies of this alga.

Characterization of H2 photoproduction by marine green alga Tetraselmis subcordiformis integrated with an alkaline fuel cell.

To investigate the feasibility of coupling carbonyl cyanide m-chlorophenylhydrazone-regulated photohydrogen production by Tetraselmis subcordiformis in a photobioreactor to an alkaline fuel cell (AFC). H2 evolution kinetics in the AFC integrated process was characterized. The duration of H2 evolution was prolonged and its yield was improved about 1.5-fold (to 78 ± 5 ml l(-1)) compared with that of the process without AFC. Improved H2 yield was possibly caused by removal of H2 feedback inhibition by H2 consumption in situ. Decreases in the H2 production rate correlated with the gradual deactivation of PSII and hydrogenase activities. The H2 yield was closely associated with catabolism of starch and protein. A marine green algal CO2-supplemented culture integrated with in situ H2-consumption by an AFC system was developed as a viable protocol for the H2 production.

Investigating Cellular Responses During Photohydrogen Production by the Marine Microalga Tetraselmis subcordiformis by Quantitative Proteome Analysis.

The marine microalga Tetraselmis subcordiformis could photoproduce hydrogen under the regulation of carbonyl cyanide m-chlorophenylhydrazone (CCCP), and a hydrogen production process kinetic analysis was characterized by two peaks, suggesting that two distinct mechanisms might exist in this alga. Therefore, 2D nanoliquid chromatography-tandem mass spectrometry (LC-MS/MS) was introduced to analyze the proteome of samples from different time points. A total of 912 proteins were identified, providing a global view of the cellular responses at the proteomic level. These proteins can be divided into multiple functional groups including stress responses, energy metabolism and redox homeostasis. The quantitative proteomic data provided more details on the electron donors for hydrogen production. During the first stage, photosystem II produced electrons for hydrogen production; during the second stage, metabolites were the major electron donors via nonphotochemical plastoquinone reduction by NADH dehydrogenase.

Temperature effects on lipid properties of microalgae Tetraselmis subcordiformis and Nannochloropsis oculata as biofuel resources

Microalgae Tetraselmis subcordiformis and Nannochloropsis oculata were cultured at 15, 20, 25, 30, and 35°C and their properties as potential biofuel resources were examined. The results indicate that T. subcordiformis and N. oculata grew best at 20°C and 25°C and yielded the highest total lipids at 20°C and 30°C, respectively. With increased temperature, neutral lipid and polyunsaturated fatty acids (FAs) decreased while saturated FAs increased, accompanied by increased monounsaturated FAs (MUFAs) in T. subcordiformis and decreased MUFAs in N. oculata; meanwhile, the predicted cetane number of FA methyl esters increased from 45.3 to 47.6 in T. subcordiformis and from 52.3 to 60.3 in N. oculata. Therefore, optimizing culture temperatures is important for improving microalgal biodiesel production.

Protein–protein interaction network of the marine microalga Tetraselmis subcordiformis: prediction and application for starch metabolism analysis

Under stressful conditions, the non-model marine microalga Tetraselmis subcordiformis can accumulate a substantial amount of starch, making it a potential feedstock for the production of fuel ethanol. Investigating the interactions of the enzymes and the regulatory factors involved in starch metabolism will provide potential genetic manipulation targets for optimising the starch productivity of T. subcordiformis. For this reason, the proteome of T. subcordiformis was utilised to predict the first protein-protein interaction (PPI) network for this marine alga based on orthologous interactions, mainly from the general PPI repositories. Different methods were introduced to evaluate the credibility of the predicted interactome, including the confidence value of each PPI pair and Pfam-based and subcellular location-based enrichment analysis. Functional subnetworks analysis suggested that the two enzymes involved in starch metabolism, starch phosphorylase and trehalose-phosphate synthase may be the potential ideal genetic engineering targets.

Application of Fourier transform infrared (FT-IR) spectroscopy in determination of microalgal compositions

Fourier transform infrared spectroscopy (FT-IR) was applied in algal strain screening and monitoring cell composition dynamics in a marine microalga Isochrysis zhangjiangensis during algal cultivation. The content of lipid, carbohydrate and protein of samples determined by traditional methods had validated the accuracy of FT-IR method. For algal screening, the band absorption ratios of lipid/amide I and carbo/amide I from FT-IR measurements allowed for the selection of Isochrysis sp. and Tetraselmis subcordiformis as the most potential lipid and carbohydrate producers, respectively. The cell composition dynamics of I. zhangjiangensis measured by FT-IR revealed the diversion of carbon allocation from protein to carbohydrate and neutral lipid when nitrogen-replete cells were subjected to nitrogen limitation. The carbo/amide I band absorption ratio had also been demonstrated to depict physiological status under nutrient stress in T. subcordiformis. FT-IR serves as a tool for the simultaneous measurement of lipid, carbohydrate, and protein content in cell.

Salinity manipulation as an effective method for enhanced starch production in the marine microalga Tetraselmis subcordiformis

Microalgal starch is considered a promising feedstock for bioethanol production. The biomass and starch accumulation in the marine microalga Tetraselmis subcordiformis were characterized under different salinities in response to nitrogen repletion (+N) or depletion (-N) at high irradiance (HI) or low irradiance (LI). Under favorable nutritional conditions (HI+N), biomass accumulation was seldom affected under 20% normal salinity, though starch accumulation were somewhat reduced. Increased salinity impaired overall biomass and starch accumulation, though it led to a temporary starch accumulation at initial cultivation phase. Under nitrogen deprivation, decreased salinity strengthened biomass and starch accumulation regardless of irradiance. The highest starch content of 58.2% dry weight and starch productivity of 0.62 g L(-1) d(-1) were obtained under HI-N with 20% normal salinity. Decreased salinity combined with -N generated moderate stress to facilitate starch accumulation. Salinity manipulation can be effectively applied for enhanced starch production in marine microalgae.

Effect of high ferric ion concentrations on total lipids and lipid characteristics of Tetraselmis subcordiformis, Nannochloropsis oculata and Pavlova viridis

Microalgae as sources for biodiesel production have been widely investigated. Microalgae biomass, lipid content and fatty acid profiles of microalgae are limiting factors for the cost-effective production of biodiesel. In this paper, the effects of high ferric ion concentrations on three species of microalgae (Tetraselmis subcordiformis, Nannochloropsis oculata and Pavlova viridis) were studied. The microalgae were cultured in different concentrations (1.2 × 10−2, 1.2 × 10−1, 1.2 and 12 mmol L−1) of ferric ion. The growth, lipid content and fatty acid profiles of the three microalgae were analysed. When algae were cultured in 1.2 mmol L−1 ferric ion for 10 days, the final cell density and specific growth rates of T. subcordiformis, N. oculata and P. viridis decreased significantly (p < 0.05), and the total lipid contents of the microalgae, 33.72, 37.34 and 29.48 % (dry mass) in T. subcordiformis, N. oculata and P. viridis, respectively, were higher than those at other concentrations. The neutral lipid/total lipid ratios of the three microalgae species increased with increasing ferric ion concentration. Neutral lipids accounted for 50.75, 48.37 and 46.59 % of the total lipid in T. subcordiformis, N. oculata and P. viridis, respectively, when cultured in 12 mmol L−1 ferric ion. The proportions of saturated fatty acids in all three species cultured in 12 mmol L−1 ferric ion were significantly higher than those cultured in lower ferric ion concentrations. An optimum ferric ion concentration can improve the properties of T. subcordiformis, N. oculata and P. viridis as sources for biodiesel.

Characterization of cell growth and starch production in the marine green microalga Tetraselmis subcordiformis under extracellular phosphorus-deprived and sequentially phosphorus-replete conditions

Microalgal starch is a potential feedstock for biofuel production. Nutrient stress is widely used to stimulate starch accumulation in microalgae. Cell growth and starch accumulation in the marine green microalga Tetraselmis subcordiformis were evaluated under extracellular phosphorus deprivation with initial cell densities (ICD) of 1.5, 3.0, 6.0, and 9.0×10⁶ cells mL⁻¹. The intracellular stored phosphorus supported cell growth when extracellular phosphorus was absent. The maximum starch content of 44.1% was achieved in the lowest ICD culture, while the maximum biomass productivity of 0.71 g L⁻¹ day⁻¹, starch concentration of 1.6 g L⁻¹, and starch productivity of 0.30 g L⁻¹ day⁻¹ were all obtained in the culture with the ICD of 3.0×10⁶ cells mL⁻¹. Appropriate ICD could be used to regulate the intracellular phosphorus concentration and maintain adequate photosynthetic activity to achieve the highest starch productivity, along with biomass and starch concentration. The recovery of phosphorus-deprived T. subcordiformis in medium containing 0.5, 1.0, or 6.0 mM KH₂PO₄ was also tested. Cell growth and starch accumulation ability could be recovered completely. A phosphorus pool in T. subcordiformis was shown to manipulate its metabolic activity under different environmental phosphorus availability. Though lower starch productivity and starch content were achieved under phosphorus deprivation compared with nitrogen- or sulfur-deprived conditions, the higher biomass and starch concentration make T. subcordiformis a good candidate for biomass and starch production under extracellular phosphorus deprivation.

Preparation of a microalgal photoanode for hydrogen production by photo-bioelectrochemical water-splitting

In this study, a microalga Tetraselmis subcordiformis (synonym: Platymonas subcordiformis)-based photoanode was prepared by a novel method developed in our lab. The optimal photocurrent density of microalgae photoanode, 37 μA/cm2, was achieved under illumination of 145 μmol s−1 m−2 at anode potential of 0.5 V vs Ag|AgCl|sat. KCl, immobilized cell density of 2.08 × 106/cm2 and BQ concentration of 300 μmol/L. The results of measurements showed that oxygen evolution peak, hydrogen evolution peak and photocurrent response were all synchronous to light impulse in a three-electrode system. It revealed that there occurred a process of photo-bioelectrochemical water-splitting. Hydrogen can be produced by the method. The investigation for whole photo-bioelectrochemical process also indicated that the electrons for hydrogen evolution had two sources, microalgal metabolic process in dark condition and photosynthetic water oxidation. The photo-hydrogen evolution was twice more than hydrogen evolution in dark condition.

Detection and quantitation of lipid in the microalga Tetraselmis subcordiformis (Wille) Butcher with BODIPY 505/515 staining

BODIPY 505/515, a lipophilic bright green fluorescent dye was tested for lipid detection in the microalga Tetraselmis subcordiformis. A concentration of 0.28μgml−1 and staining for 6min was optimal. Lipid bodies stained with BODIPY505/515 had a characteristic green fluorescence. Their volumes were determined using the sphere volume formula. Lipid accumulation under different nitrogen concentrations was analyzed. With an increase in NaNO3 concentration from 0 to 240mg L−1, the maximum algal concentration increased from 8.23±0.62 (×105 cells ml−1) to 1.61±0.13 (×106 cellsml−1), while the maximum volume of intracellular neutral lipid decreased from 9.78±1.77 μm3cell−1 to 6.00±0.59μm3cell−1. A comparison of the lipid contents measured by BODIPY 505/515 staining and the gravimetric method showed a positive correlation coefficient of R2=0.93. BODIPY 505/515 staining is a promising method in lipid quantitation in T. subcordiformis.

Enhancing starch production of a marine green microalga Tetraselmis subcordiformis through nutrient limitation

Microalgal starch is a potential feedstock for biofuel production. The effects of KNO3 and MgSO4 concentrations and light intensity on biomass and starch production by the marine microalga, Tetraselmis subcordiformis, were investigated. Under 200μmolm−2s−1 irradiance and sulfur-deprived conditions, a starch productivity of 0.62gL−1d−1 and a starch content of 62.1% based on dry weight (DW) was achieved. A starch content of 54.3% was achieved under low irradiance and nitrogen starvation, which was 6.5% higher than that under nutrient- and light-sufficient conditions. Photosynthetic activity was indispensable for starch accumulation. It is difficult to reach high starch productivity and starch concentration simultaneously. Proper nutrient concentrations are necessary to achieve high starch productivity or starch concentration based on the target. The high starch productivity and starch content suggest that T. subcordiformis is a promising microalgal starch producer.