Using bait microalga as an oral delivery vehicle of antimicrobial peptide for controlling Vibrio infection in mussels.

In shellfish aquaculture, antibiotics are commonly used to address Vibrio infections. However, antibiotic abuse has increased the risk of environment pollution, which has also raised food safety concerns. Antimicrobial peptides (AMPs) are considered safe and sustainable alternatives to antibiotics. Hence, in this study, we aimed to develop a transgenic Tetraselmis subcordiformis line harboring AMP-PisL9K22WK for reducing the use of antibiotics in mussel aquaculture. Toward this, pisL9K22WK was assembled into nuclear expression vectors of T. subcordiformis. Post particle bombardment, several stable transgenic lines were selected after 6 months of herbicide resistance culture. Subsequently, Vibrio-infected mussels (Mytilus sp.) were orally fed transgenic T. subcordiformis to test the efficacy of this drug delivery system. The results showed that the transgenic line as an oral antimicrobial agent significantly improved the resistance of mussels to Vibrio. The growth rate of the mussels fed transgenic T. subcordiformis was considerably higher than that of mussels fed wild-type algae (10.35% versus 2.44%). In addition, the possibility of using the lyophilized powder of the transgenic line as drug delivery system was also evaluated; however, compared to that observed after feeding with live cells, the lyophilized powder did not improve the low growth rate caused by Vibrio infection, suggesting that fresh microalgae are more beneficial for the delivery of the PisL9K22WK to mussels than the lyophilized powder. In summary, this is a promising step toward the development of safe and environment-friendly antimicrobial baits.

Recombinant Expression of Thrombolytic Agent Reteplase in Marine Microalga Tetraselmis subcordiformis (Chlorodendrales, Chlorophyta).

Tetraselmis subcordiformis, a unicellular marine green alga, is used widely in aquaculture as an initial feeding for fish, bivalve mollusks, penaeid shrimp larvae, and rotifers because of its rich content of amino acids and fatty acids. A stable nuclear transformation system using the herbicide phosphinothricin (PPT) as a selective reagent was established previously. In this research, the recombinant expression in T. subcordiformis was investigated by particle bombardment with the rt-PA gene that encodes the recombinant human tissue-type plasminogen activator (Reteplase), which is a thrombolytic agent for acute myocardial infarction treatment. Transgenic algal strains were selected by their resistance to PPT, and expression of rt-PA was validated by PCR, Southern blotting, and Western blotting, and bioactivity of rt-PA was confirmed by the fibrin agarose plate assay for bioactivity. The results showed that rt-PA was integrated into the genome of T. subcordiformis, and the expression product was bioactive, indicating proper post-transcriptional modification of rt-PA in T. subcordiformis. This report contributes to efforts that take advantage of marine microalgae as cell factories to prepare recombinant drugs and in establishing a characteristic pathway of oral administration in aquaculture.

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

OBJECTIVES: 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). RESULTS: 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. CONCLUSION: 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.

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.

Oral Administration of Antimicrobial Peptide NZ2114 Through the Microalgal Bait Tetraselmis subcordiformis (Wille) Butcher for Improving the Immunity and Gut Health in Turbot (Scophthalmus maximus L.).

Antibiotics are widely used in aquaculture to treat the bacterial diseases. However, the improper use of antibiotics could lead to environmental pollution and development of resistance. As a safe and eco-friendly alternative, antimicrobial peptides (AMPs) are commonly explored as therapeutic agents. In this study, a mutant strain of Tetraselmis subcordiformis containing AMP NZ2114 was developed and used as an oral drug delivery system to reduce the use of antibiotics in turbot (Scophthalmus maximus) aquaculture. The gut, kidney, and liver immune-related genes and their effects on gut digestion and bacterial communities in turbot fed with NZ2114 were evaluated in an 11-day feeding experiment. The results showed that compared with the group fed with wild-type T. subcordiformis, the group fed with T. subcordiformis transformants containing NZ2114 was revealed with decreased levels of both pro-inflammatory factors (TNF-α and IL-1ß), inhibitory effect on Staphylococcus aureus, Vibrio parahaemolyticus, and Vibrio splendidus demonstrated by the in vitro simulation experiments, and increased richness and diversity of the gut microbiota of turbot. In conclusion, our study provided a novel, beneficial, and low-cost method for controlling bacteria in turbot culture through the oral drug delivery systems.

Evidence of the drying technique's impact on the biomass quality of Tetraselmis subcordiformis (Chlorophyceae).

Rapid drying, cost-effective and safe, will increase the viability of using microalgae for several bio-industrial applications. In this study, five different drying techniques of microalgal biomass were investigated. These include freeze drying, oven drying, air drying, sun drying, and microwave drying. Morphology, metabolite content, FAME profiling, chlorophyll content, total organic carbon, and total nitrogen were analyzed. Results showed that the freeze-drying technique preserves the highest amounts of chlorophyll, proteins, and lipids. Oven drying underperformed as it retained the lowest amount of chlorophyll, protein, and lipid content. More importantly, FAME profiling results showed that air drying was the best technique in maintaining the highest amount of polyunsaturated fatty acids and more specifically docosahexaenoic acid (DHA). Furthermore, this process requires the least capital and energy needs. The findings from this study confirmed that the drying technique affects the microalga biomass quality.

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

BACKGROUND: Microalgal 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. RESULTS: The 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. CONCLUSIONS: The 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.

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 15 h of H2 evolution, the system was subjected to dark treatment after initial light illumination (L/D = 6/9 h, 9/6 h, and 12/3 h). 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 ± 10 mL L-1 was achieved using a light/dark regimen of L 9 h/D 6 h/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.

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.

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.