Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9-mediated genome editing has been used for reverse genetics studies in many organisms. However, application to commercially ...important species of seaweeds is still limited. The genetics and breeding technologies for species of the Laminariales will be advanced by developing a genome editing tool. In this study, we attempted to edit a counter-selectable marker, the
adenine phosphoribosyl transferase
(
APT
) gene using CRISPR-Cas9 ribonucleoprotein (RNP) complexes delivered by microinjection into gametophytes of
Saccharina japonica
. After injection of CRISPR-Cas9 RNP, 2-fluoroadenine (2-FA) was added to the medium (10–40 μM) to select
APT
mutants. Twenty-three female and 12 male 2-FA resistant gametophytes had mutations in their
APT
genes. Genome editing efficiency for the injection trials was 8.64% and 4.46% for the female and male gametophytes, respectively. The
apt
mutant gametophytes were able to produce sperm or eggs. Sporophytes derived by crossing sperm and eggs from
apt
mutant gametophytes were resistant to 40 μM 2-FA. Sporophytes derived from crosses with one wild type and one mutant parent also showed resistance to high concentrations (20–40 μM) of 2-FA, while wild-type sporophytes died in 10 μM 2-FA. This is the first report showing the validity of gene editing of
S. japonica
using the CRISPR-Cas9 RNP complex and microinjection method.
Ocean-based carbon dioxide removal has gained immense attention as a countermeasure against climate change. The enhancement of ocean alkalinity and the creation of new blue carbon ecosystems are ...considered effective approaches for this. To evaluate the function of steelmaking slag from the viewpoints of CO2 reduction and creation of new blue carbon ecosystems, we conducted a comparative experiment using two mesocosms that replicated tidal-flats and shallow-water ecosystems. Initially, approximately 20 seagrasses (Zostera marina) were transplanted into the shallow-water area in the mesocosm tanks. The use of steelmaking slag is expected to increase the pH by releasing calcium and mitigate turbidity by solidifying dredged soil. In the experimental tank, where dredged soil and steelmaking slag were utilized as bed materials, the pH remained higher throughout the experimental period compared with the control tank, which utilized only dredged soil. As a result, pCO2 remained consistently lower in the experimental tank due to mainly its alkaline effect (March 2019: −10 ± 6 μatm, September 2019: −130 ± 47 μatm). The light environment in the control tank deteriorated due to high turbidity, whereas the turbidity in the experimental tank remained low throughout the year. The number of seagrass shoots in the experimental tank was consistently approximately 20, which was higher than that in the control tank. Additionally, more seaweed and benthic algae were observed in the experimental tank, indicating that it was more conducive to the growth of primary producers. In conclusion, tidal-flat and shallow-water ecosystems constructed using dredged soil and steelmaking slag are expected to enhance CO2 uptake and provide a habitat for primary producers that is superior to those constructed using dredged soil only.
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•Steelmaking slag was examined as a bed material for the creation of BC ecosystems.•Alkalinity enhancement by steelmaking slag decreased pCO2 in water.•The use of steelmaking slag had no apparent negative impact on primary producers.
The present study investigated the monthly of element accumulation in seaweeds. Patterns of As, Ba, Cd, Cu, Fe, Mn, Pb, and Zn concentrations in dominant species of Phaeophyceae and Rhodophyceae, ...namely Saccharina japonica and Pterocladiella tenuis respectively, collected from the Betsukari coastline-Mashike, Hokkaido, Japan, were investigated. Our results indicated that element accumulation was more related to specific seaweed species than to their supply in seawater concentration. S. japonica was found to be an accumulator of As, whereas P. tenuis notably accumulated Mn. The accumulation of specific elements also affects the coupled patterns between closely related elements. The monthly pattern of Cd was similar to that of As in S. japonica, and it is an element with unknown biological function in the seaweed. The monthly accumulation pattern of Fe and Mn, a well-known closely related element that forms the extracellular surface in seaweed, was found to be similar in P. tenuis. A similar transport mechanism affected the antagonistic pattern of Cd and Zn accumulation in S. japonica. Our data can be employed in the assessment of biomonitoring of element cycles in the environment.
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•Monthly changes in element accumulation in two seaweed species were studied.•S. japonica and P. tenuis accumulate prominent As and Mn, respectively.•Surface bound concentration of Fe and Mn is higher than intracellular accumulation.•Intracellular accumulation of As and Cd increases monthly.•Similar active transport in seaweed led to discrete pattern of Cd & Zn accumulation.
Macroalgae can cycle arsenic (As) in the environment. In this study, the role of iron (Fe) plaque manipulation at active sites in the As biotransformation mechanism was investigated. The strain of ...marine macroalgal species, Pyrophia yezoensis, was inoculated in association with arsenate (As(V)) (1.0 μmol L−1) and phosphate (10 μmol L−1) in the medium for 7 days under laboratory-controlled conditions. The Fe plaque was removed by washing the Ti(III)-citrate-EDTA solution before inoculation. The limitation of Fe plaque did not significantly (p > 0.05) affect the chlorophyll fluorescence due to cellular regeneration, which was initiated immediately after washing. However, the speciation and uptake rate of As(V) increased significantly and reduced the inhibitory effect of P on the intracellular uptake of As(V) by P. yezoensis. In the culture medium without Fe plaque, approximately 66% of As(V) was removed with Vmax = 0.32 and Km = 1.92. In the absence of Fe plaque, methylated As species, such as dimethylarsinate (DMAA(V)), was recorded 0.28 μmol L−1, while in the presence of Fe plaque, the value was 0.16 μmol L−1. Inorganic trivalent As (As(III)) was absent in the washed samples; however, 0.53 μmol L−1 concentration of As(III) was still found in the presence of Fe plaque on day 7 of incubation. The results indicated that the absence of Fe plaque promoted higher intracellular uptake of As species, reduced the inhibitory effect of P, mitigated the co-precipitation bond between AsFe plaque and enhanced the detoxification process by DMAA excretion from the cell.
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•Fe plaque role on As biotransformation by marine macroalgae is investigated.•Methylation of As species influenced by the concentrations of As(V) and P in the medium.•Absence of Fe plaque promoted higher intracellular uptake of As species.•Competition between As(V) and P decreases without Fe plaque.•Presence of Fe plaque affect the detoxification process by DMAA excretion from the cell.
The toxicity and bioaccumulation and biotransformation potential of inorganic arsenic (IAs) species As(V) and As(III) were investigated using
Sargassum patens
under laboratory culture for 7 days. ...Algal chlorophyll fluorescence decreased with increasing As(V) and As(III) concentrations, being significantly affected by As(III) treatments. Higher As(III) concentration negatively affected growth rate, and P and Fe limitation greatly enhanced IAs toxicity. The extracellular, intracellular, and total bioaccumulation of As(III) and As(V) varied significantly depending on initial concentrations and addition of P and Fe. P and Fe availability suppressed intracellular As accumulation in As(V) medium but not in As(III) medium. In P-rich (10 μmol L
−1
) medium, intracellular As was reduced by 4.7% and 9.9% when As(V) in the medium was constant (4.0 μmol L
−1
), under Fe-limited (0 μmol L
−1
) and Fe-rich (10 μmol L
−1
) conditions, respectively. However, the Fe-rich condition positively affected extracellular As accumulation from both As source. Extracellular As increased by 43.5% and 38.8% in P-limited + Fe-rich cultures with 4.0 μmol L
−1
of As(V) and As(III), respectively. Algae exhibited greater absorption and adsorption to As(V) than to As(III). The reduced metabolites of As(III) (3.5 to 4.9% of the total As) and oxidized metabolites of As(V) (2.0 to 3.7% of the total As) were recorded as biotransformed species from coexisting media containing As(V) and As(III) at a constant 4.0 μmol L
−1
, respectively. Both P and Fe had significant influences on the variation in behaviors of IAs. This information is vital in terms of As research in marine ecosystems.
Freshwater and marine organisms are capable of metabolizing arsenic (As) efficiently and regulating the As biogeochemical cycles. In this study, Undaria pinnatifida was exposed to As(V) (0, 0.1, and ...1 μM) and phosphate (P; 1 and 10 μM) in seawater under laboratory-controlled conditions for up to seven days to analyze As biotransformation. The growth rates and chlorophyll fluorescence of the alga were unaffected by As stress, and statistically insignificant differences were observed among the cultures (p > 0.05). As(V) was readily accumulated by this macroalga through phosphate transporters, transformed intracellularly, and excreted into the medium, depending on the As(V) to P molar ratios. The concentration of As(V) and biotransformed species As(III) and DMAA(V) varied significantly in the algal cultures on the basis of the exposure period (p < 0.05). The concentration of As(III) was initially higher but decreased with the incubation period, whereas the concentration of DMAA(V) increased gradually. At the end of the incubation, 0.04 and 0.32 μM DMAA(V) were recorded in the media containing 0.1 and 1 μM As(V) with a constant 1.0 μM P, respectively. The results also indicated that the cellular uptake of As(V) and subsequent release of DMAA(V) were inhibited by P in the medium. The biotransformation was consistent with the As(V) detoxification mechanism based on reduction and methylation, which was enhanced by the lower As(V) to P molar ratios. These findings can be helpful in understanding the contribution of macroalgae to As biogeochemistry in marine environments and the potential risks of As dietary intake.
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•The growth rate was significantly unchanged up to seven days with 1.0 μM of As(V).•The overall biotransformation of As(V) was coupled with reduction and methylation.•As(V) uptake and its biotransformation was enhanced by low levels of P in culture.•The intracellular accumulation of As was minimum at initial high levels of P.•The presence of Fe in medium can lead to adsorb As and P on the surface of algae.
To aid in the restoration of coastal barren ground areas, it is important to clarify the effects of chelated iron on the growth of seaweed. In particular, for the further development of practical ...methods to promote seaweed growth, Fe-binding organic ligands, such as humic substances (HSs) composed of humus materials, rather than Fe-binding inorganic ligands, such as ethylenediaminetetraacetic acid (EDTA), should be investigated. In this study, the effects of an Fe fertilizer, made from HSs and steelmaking slag, on the growth of the brown alga
Sargassum horneri
at the germling and immature stages were examined. The addition of the Fe fertilizer eluate containing Fe organic ligand complexes clearly promoted the growth of
S. horneri
at the germling and immature stages. It was also clear that the effect of Fe concentration in the Fe fertilizer eluate on the growth rate was almost the same as that of Fe–EDTA. Moreover, the addition of the Fe fertilizer eluate had a great effect on the brown color of
S. horneri
thalli and promoted the increased content of photosynthetic pigments, such as chlorophyll
a
. Based on these experimental results, the application of the Fe fertilizer containing Fe organic ligand complexes is expected to become an effective method for the restoration of the barren ground phenomenon in Fe-deficient coastal areas.
Biological treatments using microalgae represent an environmental-friendly approach to the decontamination of coking wastewater, which contains toxic levels of ammonium; however, few algal species ...can tolerate such high ammonium levels. In this study, we investigated the morphology, ultrastructure, molecular phylogeny, and physiological characteristics of Parachlorella kimitsuensis sp. nov., a new algal species with high ammonium tolerance. This species was isolated from coking wastewater obtained near a steelworks factory, where the ratio of nitrogen to phosphorus (N/P) exceeded 40, approximately 100-fold that of general culture medium. The growth inhibition test estimated that the effective concentration at 10 % inhibition (EC10) was 3.2 ppm NH4+-N, and the half maximal effective concentration (EC50) was 98.0 ppm NH4+-N, indicating tolerance to fairly high concentrations of ammonium in this species. Parachlorella kimitsuensis has spherical, green cells resembling those of Chlorella species. The major diameter of the cells was 3.7 ± 0.39 μm; cells containing eight autospores appearing in the early logarithmic growth phase were 6.9 ± 0.78 μm in diameter. Molecular phylogenetic analysis showed that the alga was a member of the genus Parachlorella, with 100 % bootstrap support. However, the results of vegetative cell morphology and phylogenetic analyses using 18S rRNA genes were inconsistent with those of other described Parachlorella species. Therefore, we identified the isolate as a new species in the genus Parachlorella. We anticipate that this species will be suitable for biological treatment of wastewater containing high ammonium levels, such as coking wastewater.
•A new strain of Parachlorella isolated from coking wastewater•Adapted to highly ammonium and salinity of seawater•Potential for biological treatment of wastewater containing ammonium
Pyrolysis of marine biomass (MB) to create marine biomass-derived carbon materials (MBCs) for industry is a renewable solution. In this study, we systematically examined pyrolysis processes and ...performed elemental composition analyses of MB and MBC. The results revealed that MBC contained 12.5–25.5 mass% of alkali and alkaline earth metals (AAEMs). As the pyrolysis temperature increased, some of these AAEMs underwent physical phase changes. Subsequently, we applied CO2 aeration washing for the first time or acetic acid washing to eliminate AAEMs from MBC, reducing potential hazards in the future industrial application.
Characterization results suggested that both post-processes effectively removed AAEMs from MBC, reducing the average content of Na and K to 0 mass%, Ca to 1.4 mass%, and Mg to 1.9 mass%. The Cl content was also reduced to 0.1 mass%. After removing the AAEMs, the final carbon content of the MBCs according to ultimate analysis was as high as 90.8 mass% (dry ash free basis) with a minimum ash content of 6.4 mass% (dry basis). Comparison with commercial coal products showed that carbon contents were superior and ash contents were lower than those of most coal samples, suggesting that the treated MBCs are of sufficient quality for the carbon cycle industry.
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