Helgolandica Var Research Articles

Transcriptomics reveal how circadian regulation contributes to starch hyperaccumulation in marine alga Tetraselmis helgolandica

AbstractTetraselmis helgolandica var. Tsingtaoensis is a marine microalga. It can produce a large amount of starch, especially amylose, with addition of carbon source and specific circadian rhythm. The mechanism behind this phenomenon is still unclear. Analysis of this mechanism can help to develop T. helgolandica into a new green bioengineering chassis organism. We explained how circadian rhythm and glucose affect the rate of starch accumulation and starch structure in T. helgolandica based on the transcriptome. The glucose inhibited the photosynthetic system of T. helgolandica, while the circadian rhythm can alleviate the inhibition. Circadian rhythm induced the upregulation of Embden–Meyerhof–Parnas pathway and pentose phosphate pathway (PPP) in T. helgolandica, but had little effect on the tricarboxylic acid cycle. PPP pathway provides Ribulose‐1,5‐bisphosphate, which may be beneficial for dark reactions and nucleotide synthesis. And PPP pathway provides Nicotinamide adenine dinucleotide phosphate, which facilitates energy substance synthesis. This will further upregulate the starch metabolic pathway. The transcript level of the key gene ADP‐Glucose pyrophosphorylase is mainly regulated by glucose. The granule‐bound starch synthase (gbss), a key gene for amylose synthesis, is mainly influenced by circadian rhythm. In general, the increase of starch synthesis and amylose ratio requires both glucose addition and circadian rhythm. We report the first referenced transcriptome of T. helgolandica. Differences between transcripts reveal how circadian rhythm and glucose addition affected the rate of starch synthesis and structural variation. It provides a reference for an in‐depth study of starch synthesis in green algae.

Platymonas helgolandica-driven nitrogen removal from mariculture wastewater under different photoperiods: Performance evaluation, enzyme activity and transcriptional response

The nitrogen removal performance and biological mechanism of Platymonas helgolandica var. Tsingtaoensis (P. helgolandica) were investigated in treating mariculture wastewater under different light: dark (L:D) photoperiods. The growth of P. helgolandica was positively correlated with the photoperiods from 6L:18D to 15L:9D, and the highest photosynthetic activity appeared under 6L:18D photoperiod on day 3. P. helgolandica exhibited the highest removal efficiencies of total nitrogen and COD at 89 % and 93 % under 15L:9D photoperiod, respectively. NH4+-N assimilation was proportional to the photoperiods from 6L:18D to 15L:9D and longer illumination promoted NO2−-N removal. However, the highest NO3−-N reduction rate was achieved under 12L:12D photoperiod. The different nitrogen-transformed enzymatic activities were affected by photoperiod. Transcriptome revealed that unigenes were enriched in nitrogen metabolism and photosynthesis pathways, of which the functional gene expression was up-regulated significantly. This study provides insights into the optimization of photoperiod for mariculture wastewater treatment by P. helgolandica.

Polystyrene nanoplastics alleviate the toxicity of CuO nanoparticles to the marine algae Platymonas helgolandica var. tsingtaoensis

The increasing presence of pollutants such as engineered nanoparticles (ENPs) and nanoplastics (NPLs) poses potential risks to the marine environment. However, there is a scarcity of information on their joint toxic effects. In this study, we investigated the toxicity of copper oxide nanoparticles (nCuO) combined with polystyrene nanoplastics (PS-NPLs) on the marine microalgae Platymonas helgolandica var. tsingtaoensis. The inhibitory effect of nCuO increased with increasing concentrations, and nCuO ≥ 100 mg/L significantly restrained the growth and chlorophyll content of microalgae. nCuO could be adsorbed by algal cells, which was responsible for membrane lipid oxidation and the disruption of membrane permeability. Simultaneous exposure to nCuO and PS-NPLs had a.n antagonistic effect on the growth inhibition of the microalgae, and nCuO played a leading role in the joint toxicity of nCuO and PS-NPLs. In comparison to nCuO exposure alone, combined exposure decreased the oxidative stress and alleviated the increase in the cell membrane permeability of microalgae. PS-NPLs could heteroaggregate with nCuO, which reduced the interaction between nCuO and microalgae, inducing decreased joint toxicity. Findings of this study will clarify our understanding of the joint toxicity of ENPs and NPLs.

A microfluidic droplet array demonstrating high-throughput screening in individual lipid-producing microalgae

Microalgae are a group of photoautotrophic microorganisms which could use carbon dioxide for autosynthesis. They have been envisioned as one of the most prospective feedstock for renewable oil. However, great endeavors will still be needed to increase their economic feasibility. The screening of competitive species and suitable culture conditions are such issues. To greatly accelerate these rather laborious steps and also improve their experimental lump-sum-manner, we developed a microfluidic droplet-based 2 × 103 resolution “identification card”, which allowed high throughput real-time monitoring of individual algae among population. A novel fluid-blocking-based droplet generating and trapping performance were integrated in the platform which made it excellent in operational simplicity, rapidity and stability and full of the potentials in single-cell-isolation/screening. The developed platform was successfully used to screen three unicellular algae, namely, Isochrysis zhanjiangensis, Platymonas subcordiformis and Platymonas helgolandica var. tsingtaoensis. In situ bioassays of the lipid accumulation and cell proliferation at single cell level for interspecies comparison were possible. Furthermore, lipid-producing inhomogeneity was demonstrated among cells in the same specie and batch. Nitrogen stress condition can be identified that induce positive-skewed frequency distribution of lipid content, even among individual inhomogeneous cells over the typically used culture condition.

Single and combined toxicity of polystyrene nanoplastics and copper on Platymonas helgolandica var. tsingtaoensis: Perspectives from growth inhibition, chlorophyll content and oxidative stress

The combined toxic effects of nanoplastics and heavy metals on aquatic organisms have attracted widespread attention; however, the results are inconsistent and the mechanisms remain unclear. In this study, the single and combined toxicity effects of Cu and two types of polystyrene nanoplastics (PS-NPs; 50 nm PS and 55 nm PS-COOH) on Platymonas helgolandica var. tsingtaoensis were investigated, including growth inhibition, chlorophyll content, and oxidative stress. An adverse dose-response relationship on growth inhibition was found in the Cu treatment groups, which was related to the decrease in chlorophyll content and damage to cell membranes. The growth inhibitory effect of PS-NPs on microalgae increased with exposure time and concentration, and no significant difference was found in the two types of PS-NPs because of the negligible contribution of functional groups. A more significant increase in chlorophyll content was found in PS treatments than in PS-COOH treatments at 96 h because of the microscale aggregates formed by PS. Higher concentrations (≥ 50 mg/L) of PS-NPs caused membrane lipid peroxidation, which might be responsible for growth inhibition. In the combined exposure experiments, a synergistic effect on the growth inhibition rate was obtained using the independent action model and Abbott model. Combined exposure triggered more severe oxidative damage to the microalgae. Adsorption experiment results showed that there was no adsorption between PS-NPs and Cu, while the interaction of Cu and algal cells could be promoted due to the presence of the PS-NPs, which explained the increasing combined toxicity. This study could improve our understanding of the combined toxicity of nanoplastics and heavy metals and could provide a new explanation for the mechanism of combined toxicity.

Size-dependent cellular internalization and effects of polystyrene microplastics in microalgae P. helgolandica var. tsingtaoensis and S. quadricauda

Microplastics (MPs) are persistent contaminants in aquatic environments. Microalgae, as the main phytoplankton and primary producers, usually co-exist with MPs. Despite previous studies that have proved the interaction of MPs and microalgae, it is largely unknown whether MPs can be uptake into cells of microalgae. In this study, both marine P. helgolandica var. tsingtaoensis and freshwater microalgae S. quadricauda were respectively exposed to 10 mg/L polystyrene microbeads with five diameter sizes: 1.0, 2.0, 3.0, 4.0, and 5.0 μm. Confocal laser scanning and 3D image analysis showed that mean 24.0 % or 11.3 % cells of P. helgolandica var. tsingtaoensis contained 1.0 μm or 2.0 μm MPs after 72 h exposure. While mean 43.3 % or 15.3 % of S. quadricauda individuals engulfed 1.0 μm or 2.0 μm MPs within cells. But, none of 3.0–5.0 μm MPs were observed within algal cells. These results demonstrate the size-dependent cellular internalization of MPs in microalgae. Exposure to 1.0–2.0 μm PS MPs caused a significant reduction in the density of microalgae and influenced photosynthesis, which suggests cellular internalization of MPs can influence algal fertility and growth. This discovery first confirms cellular internalization of MPs in phytoplankton, of significance for the fate and eco-toxicity of MPs in the aquatic ecosystem.

A toxic effect at molecular level can be expressed at community level: A case study on toxic hierarchy

This study demonstrated hierarchical toxicity and addressed the relevance and differences of toxic effects at the molecular, individual, population, and community levels. Superoxide dismutase (SOD) activity, photosynthetic oxygen production, filtration rate, life span and densities of Platymonas helgolandica var. tsingtaoensis, Isochrysis galbana, and Brachionus plicatilis in single-species tests and customized community tests were examined in response to a concentration gradient of aniline ranging from 0 to 50.0 mg L−1. The SOD activity was the most sensitive endpoint with the fastest response to aniline according to the calculated no-detection of toxic effect concentration (NDEC) and the EC50. The individual- and population-level endpoints, showing a lower response to aniline, could be constructed from the SOD activity in a stepwise manner. A multi-scale hierarchical model with endpoints at 4 levels was used to characterize toxic effects, at the scales of time and size. Linkage of SOD activity to toxic effects at a community level was established level by level to express the change in the customized community with the concentration of aniline. The calculated threshold concentration of aniline for the customized community was nearly equal to the minimum NDEC, demonstrating as great an impact on interactions by the toxic effect at subpopulation-level as that at the community level. However, we identified a trend of higher sensitivities of measured endpoints at sub-population level, decreasing sensitivity at higher levels but a great variety of sensitivities at community level. Although the characteristics of toxic effects are different at different levels, the structure and process of endpoints at adjacent levels are related to and interact with each other. The resulted indirect effects, together with direct effect, determine the toxic effect at every levels of biological complexity. The toxic effects at adjacent levels should be studied at the same time to better understand the ecological risk of contaminants.

Elevated Seawater Temperatures Decrease Microbial Diversity in the Gut of Mytilus coruscus.

The gut microbial community is critical for the host immune system, and in recent years, it has been extensively studied in vertebrates using ‘omic’ technologies. In contrast, knowledge about how the interactions between water temperature and diet affect the gut microbiota of marine invertebrates that do not thermoregulate is much less studied. In the present study, the effect of elevated seawater temperature and diet (Isochrysis zhanjiangensis and Platymonas helgolandica var. tsingtaoensis) on the gut microbial community of the commercial mussel, Mytilus coruscus, was investigated. The 16S rRNA gene sequencing was used to characterize the microbial community in M. coruscus gut. The mortality of M. coruscus exposed to a high water temperature (31°C) increased after 3 days and the diversity of the bacterial community in the gut of live M. coruscus was significantly reduced. For example, the abundance of Bacteroides (Bacteroidetes) and norank_Marinilabiaceae (Bacteroidetes) increased in the gut of M. coruscus fed I. zhanjiangensis. In M. coruscus fed P. helgolandica, the abundance of Arcobacter (Proteobacteria) and norank_Marinilabiaceae increased and the abundance of unclassified_Flavobacteriaceae (Bacteroidetes) decreased. The results obtained in the present study suggest that high temperatures favored the proliferation of opportunistic bacteria, including Bacteroides and Arcobacter, which may increase host susceptibility to disease. Microbial community composition of the gut in live M. coruscus was not impacted by the microalgal diet but it was modified in the group of mussels that died. The present study provides insight into the potential effects on the gut microbiome and mussel–bacteria interactions of rising seawater temperatures.

The effect of naphthenic acids on physiological characteristics of the microalgae Phaeodactylum tricornutum and Platymonas helgolandica var. tsingtaoensis

Naphthenic acids (NAs) account for 1–2% of crude oil and represent its main acidic component. However, the aquatoxic effects of NAs on marine phytoplankton and their ecological risks have remained largely unknown. Using the marine microalgae Phaeodactylum tricornutum and Platymonas helgolandica var. tsingtaoensis as the target, we studied the effects of NAs on their growth, cell morphology and physiological characteristics. The cell density decreased as the concentrations of NAs increased, indicating that they had an adverse effect on growth of the investigated algae in a concentration-dependent manner. Moreover, scanning electron microscopy revealed NAs exposure caused damage such as deformed cells, shrunken surface and ruptured cell structures. Exposure to NAs at higher concentrations for 48 h significantly increased the content of chlorophyll (Chl) a and b in P. tricornutum, but decreased their levels in P. helgolandica var. tsingtaoensis. NAs with concentrations no higher than 4 mg/L gradually enhanced the Chl fluorescence (ChlF) parameters and decreased the ChlF parameters at higher concentrations for the two marine microalgae. Additionally, NAs induced hormesis on photosynthetic efficiency of the two microalgae and also have the species difference in their aquatic toxicity. Overall, the results of this study provide a better understanding of the physiological responses of phytoplankton and will enable better risk assessments of NAs.

The ecological competition and grazing reverse the effects of sulfamethoxazole on plankton: a case study on characterizing community-level effect.

The toxic effects of sulfamethoxazole (SMX) on densities of two algae, Platymonas helgolandica var. tsingtaoensis, Isochrysis galbana, and of a rotifer, Brachionus plicatilis, were tested by the population and community experiments. Two endpoints, the carrying capacity and the densities array of community in steady state (DACS), were used to characterize the toxic effects at a population level and a community one, respectively. The results showed that the carrying capacity of P. helgolandica var. tsingtaoensis and B. plicatilis did not decline significantly in population test when the concentration of SMX was lower than 6.0mgL-1 and 12.0mgL-1, respectively. However, I. galbana was sensitive to SMX and had presented toxic effect at 3.0mgL-1. By extrapolation of toxic effect at a population level to a community one, a derived community-NOEC was 3.0mgL-1, representing an inference from data of toxic effects at population level. In community experiment, when the customized community was in steady state, the density of I. galbana increased as a whole with SMX concentration in the range of tested concentration (0-144mgL-1), while that of P. helgolandica var. tsingtaoensis assumed the trend of a reversed "v" in this range. Only the density of B. plicatilis decreased with SMX concentration. With the DACS as endpoint, a NOEC for the customized community was determined to be 6.0mgL-1. This indicates that interspecific interactions can reverse the toxic effects of SMX on phytoplankton. The DACS was reliable and stable, serving as the endpoints in assessment of the effects of the pollutants on the ecosystems.

A method linking the toxic effects at community-level with contaminant concentrations

In this study, we developed a method to quantify and link the toxic effects in community-level ecosystems with concentrations of petroleum hydrocarbons. The densities of Platymonas helgolandica var. tsingtaoensis, Isochrysis galbana, and Brachionus plicatilis in single-species tests and customized ecosystems were examined in response to a concentration gradient of petroleum hydrocarbons ranging from 0 to 8.0mgL−1. A three-population ecological model with interspecies competition-grazing relationships was used to characterize population sizes with concentrations of petroleum hydrocarbons. A threshold concentration of the simplified plankton ecosystem of 0.376mgL−1 for petroleum hydrocarbons was calculated from the proposed model, which was higher than the no-effect concentration of 0.056mgL−1 derived from the single-species toxicity tests and the predicted no-effect concentration of 0.076mgL−1 calculated from the species sensitivity distribution. This finding indicates that interspecies competition and grazing reduced the toxic effect of petroleum hydrocarbons at the community level. The sensitivity analysis for model parameters demonstrates that plankton population biomasses are highly sensitive to filtration rates. Antagonism between interspecies interactions and petroleum hydrocarbon toxicity was attributed to the reduced filtration rate and zooplankton grazing pressure. The proposed method is a simple means to address the concern regarding the impacts of ecological interactions on ecological risk assessments of pollutants.

The Effects of Petroleum Hydrocarbons on Algae Can Be Reversed in the Presence of a Primary Consumer.

The ecotoxicological effects of a mixture of petroleum hydrocarbons were tested on densities of two algae, Platymonas helgolandica var. tsingtaoensis and Isochrysis galbana, and of a rotifer, Brachionus plicatilis, by single-species and customized community experiments. Test concentrations ranged from 0 to 100 mg L(-1), while five to seven treatments were assessed in triplicate within 1 month. A significant decrease in densities during single-species toxicity tests were found when concentrations of petroleum hydrocarbons were above 1.0 mg L(-1). However, equilibrium densities of algae in the customized community showed a different pattern, which increased with concentration and reached a peak at 20.0 mg L(-1). The community-based no observed effect concentration (NOEC; 1.0 mg L(-1)) was different from the NOEC derived by single-species toxic tests (0.25 mg L(-1)). This demonstrates that ecotoxicological effects on plankton as part of a community is significantly different from single-species toxicity tests owing to ecological interactions.

Effect of anthracene on the interaction between Platymonas helgolandica var. tsingtaoensis and Heterosigma akashiwo in laboratory cultures

Two species of marine phytoplankton, Platymonas helgolandica var. tsingtaoensis and Heterosigma akashiwo, were cultivated in bi-algal cultures to investigate the effect of anthracene (ANT) on the interaction between them. Without ANT, H. akashiwo out-competed P. helgolandica at low initial biomass ratios (P. helgolandica (P): H. akashiwo (H) = 1:4 and 1:1), but not at the highest (P:H = 4:1). This observation was consistent with the description in Lotka-Volterra two species competition model. It was found that P. helgolandica was excluded at low initial biomass ratios, while the unstable equilibrium between two species was predicted at the highest. For both species, carrying capacity and maximal specific growth rate decreased in bi-algal cultures compared to those in monocultures. H. akashiwo exhibited a higher sensitivity to ANT than P. helgolandica. This resulted markedly in a reduced cell density of H. akashiwo but an increased cell density of P. helgolandica. Carrying capacity of P. helgolandica was consistently higher in bi-algal cultures with ANT than those without ANT, suggesting that ANT, through the elimination of H. akashiwo, generated the dominance of P. helgolandica independently of initial biomass ratios. This study showed a density-dependent effect of harmful alga (H. akashiwo) on dietary alga (P. helgolandica), and indicated that ocean pollutant ANT could induce the succession of marine phytoplankton.

Microalgal motility measurement microfluidic chip for toxicity assessment of heavy metals

A polydimethylsiloxane microfluidic chip has been developed for the estimation of toxic heavy metals based on measurement of mobility of marine microalgae. The chip is mainly composed of an upstream concentration gradient generator and a downstream perfusion-based chemotatic module. The processes of toxic liquid dilution and diffusion, microalgal culturing, cell stimulation, and online screening can be integrated in this chip, which makes it an attractive approach to simplify toxicity testing procedures. The microalgal motility was adopted as a microfluidic bioassay signal and was evaluated as the percentage of motile cells, curvilinear velocity, average path velocity, and straight line velocity. Two mobile marine microalgae, Platymonas subcordiformis and Platymonas helgolandica var. tsingtaoensis, were confined in the chemotatic module and stimulated by the eight concentration gradients of Cu and Cd generated by the concentration gradient generator. In all cases, a toxic response was detected (i.e., a dose-related inhibition of motility was observed). Only 1.5 h was needed to predict EC(50) values. Thus, the microfluidic chip developed was proved to be useful as a simple and rapid approach in heavy metal detection and might be expanded as a conventional test method in environmental toxicity assessment.

Characterization of H 2 photoproduction by a new marine green alga, Platymonas helgolandica var. tsingtaoensis

A significant improvement in H 2 photoproduction was observed in the marine green alga Platymonas helgolandica var. tsingtaoensis in the presence of the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP). The addition of 15 μM CCCP to algal cultures led to a 260-fold increase in H 2 production compared with a negligible volume of H 2 gas produced without CCCP. The photosystem (PS) II photochemical activity was shown to be completely inhibited in the presence of CCCP under illumination. The added herbicide 3-(3,4-dichlorophenyl)-1, 1-dimethylurea further resulted in a twofold increase in H 2 photoproduction in the CCCP-treated cultures. These results demonstrated the absence of PSII-dependent pathway of H 2 photoproduction. H 2 production essentially depends on electrons donated to the hydrogenase by the endogenous substrate catabolism in CCCP-treated P. helgolandica var. tsingtaoensis. An analysis of the O 2-tolerance of the hydrogenase of P. helgolandica var. tsingtaoensis suggested that this enzyme may belong to the class [FeFe]-hydrogenase.

Laboratory study on the ecological impact of sophorolipid used for harmful algae elimination

We studied the role of sophorolipid in inhibiting harmful algae bloom (HAB). Different sophorolipid concentrations were tested on marine microalgae, zooplankton, fish, and bivalve (Mytilus edulis) in laboratory. The result shows that sophorolipid could inhibit the growth of algal species selectively. Among three algae species selected, Platymonas helgolandica var. tsingtaoensis was promoted with increasing sophorolipid concentration; Isochrysis galbana was inhibited seven days later in sophorolipid concentration below 40 mg/L; and Nitzschia closterium f. minutissima was inhibited obviously in only a high sophorolipid concentration over 20 mg/L. Therefore, sophorolipid in a low concentration at <20 mg/L could remove certain harmful algae species effectively without harming other non-harmful microalgae. For other animals, sophorolipid could inhibit the growth of ciliate Strombidium sp. by 50% at 20 mg/L sophorolipid concentration after 96 h. The concentration in 96-h LC50 for Calanus sinicus, Neomysis awatschensis, Lateolabrax japonicus, and Paralichthys olivaceus was 15, 150, 60, and 110 mg/L, respectively. The 24 h LC50 value for Artemia salina was 600 mg/L. The relative clearance rate of mussel Mytilus edulis decreased to 80%, 40%, and 20% of the control group after being exposed to 20, 50, and 100 mg/L sophorolipid for 24 h. Therefore, the toxicity for mitigation of harmful algae bloom at previously recommended concentration of 5–20 mg/L sophorolipid is low for most tested organisms in this reaserch.