De-eutrophication of effluent wastewater from fish aquaculture by using marine green alga Ulva pertusa

The de-eutrophication abilities and characteristics of Ulva pertusa, a marine green alga, were investigated in Qingdao Yihai Hatchery Center from spring to summer in 2005 by analyzing the dynamic changes in NH ₄ ⁺ , NO ₃ ^? , NO ₂ ^? as well as the total dissolved inorganic nitrogen (DIN). The results show that the effluent wastewater produced by fish aquaculture had typical eutrophication levels with an average of 34.3 μmol L^?1 DIN. This level far exceeded the level IV quality of the national seawater standard and could easily lead to phytoplankton blooms in nature if discarded with no treatment. The de-eutrophication abilities of U. pertusa varied greatly and depended mainly on the original eutrophic level the U. pertusa material was derived from. U. pertusa used to living in low DIN conditions had poor DIN removal abilities, while materials cultured in DIN-enriched seawater showed strong de-eutrophication abilities. In other words, the de-eutrophication ability of U. pertusa was evidently induced by high DIN levels. The de-eutrophication capacity of U. pertusa seemed to also be light dependent, because it was weaker in darkness than under illumination. However, no further improvement in the de-eutrophication capacity of U. pertusa was observed once the light intensity exceeded 300 μmol M² S^?1. Results of semi-continuous wastewater replacement experiments showed that U. pertusa permanently absorbed nutrients from eutrophicated wastewater at a mean rate of 299 mg/kg fresh weight per day (126 mg/kg DIN during the night, 173 mg/kg in daytime). Based on the above results, engineered de-eutrophication of wastewater by using a U. pertusa filter system seems feasible. The algal quantity required to purify all the eutrophicated outflow wastewater from the Qingdao Yihai Hatchery Center into oligotrophic level I clean seawater was also estimated using the daily discharged wastewater, the average DIN concentration released and the de-eutrophication capacity of U. pertusa.     

Screening and isolation of the algicidal compounds from marine green alga Ulva intestinalis

Twenty species of seaweed were collected from the coast of Zhejiang, China, extracted with ethanol, and screened for algicidal activity against red tide microalgae H eterosigma akashiwo and Prorocentrum micans. Inhibitory eff ects of fresh and dried tissues of green alga U lva intestinalis were assessed and the main algicidal compounds were isolated, purifi ed, and identifi ed. Five seaweed species, U. intestinalis, U. fasciata, Grateloupia romosissima, Chondria crassicaulis, and Gracilariopsis lemaneiformis, were investigated for their algicidal activities. Fresh tissues of 8.0 and 16.0 mg/m L of U. intestinalis dissolved in media signifi cantly inhibited growth of H. akashiwo and P. micans, respectively. Dried tissue and ethyl acetate(Et OAc) extracts of U. intestinalis at greater than 1.2 and 0.04 mg/m L, respectively, were fatal to H. akashiwo, while its water and Et OAc extracts in excess of 0.96 and 0.32 mg/m L, respectively, were lethal to P. micans. Three algicidal compounds in the Et OAc extracts were identifi ed as 15-ethoxy-(6z,9z,12z)-hexadecatrienoic acid(I),(6E,9E,12E)-(2-acetoxy- β- D-glucose)-octadecatrienoic acid ester(II) and hexadecanoic acid(III). Of these, compound II displayed the most potent algicidal activity with IC_(50) values of 4.9 and 14.1 μg/m L for H. akashiwo and P. micans, respectively. Compound I showed moderate algicidal activity with IC_(50) values of 13.4 and 24.7 μg/m L for H. akashiwo and P. micans, respectively. These fi ndings suggested that certain macroalgae or products therefrom could be used as ef fective biological control agents against red tide algae.     

Screening and isolation of the algicidal compounds from marine green alga <Emphasis Type="Italic">Ulva intestinalis</Emphasis>

Twenty species of seaweed were collected from the coast of Zhejiang, China, extracted with ethanol, and screened for algicidal activity against red tide microalgae Heterosigma akashiwo and Prorocentrum micans. Inhibitory effects of fresh and dried tißsues of green alga Ulva intestinalis were assessed and the main algicidal compounds were isolated, purified, and identified. Five seaweed species, U. intestinalis, U. fasciata, Grateloupia romosissima, Chondria crassicaulis, and Gracilariopsis lemaneiformis, were investigated for their algicidal activities. Fresh tissues of 8.0 and 16.0 mg/mL of U. intestinalis dissolved in media significantly inhibited growth of H. akashiwo and P. micans, respectively. Dried tissue and ethyl acetate (EtOAc) extracts of U. intestinalis at greater than 1.2 and 0.04 mg/mL, respectively, were fatal to H. akashiwo, while its water and EtOAc extracts in excess of 0.96 and 0.32 mg/mL, respectively, were lethal to P. micans. Three algicidal compounds in the EtOAc extracts were identified as 15-ethoxy-(6z,9z,12z)-hexadecatrienoic acid (I), (6E,9E,12E)-(2-acetoxy-β-D-glucose)-octadecatrienoic acid ester (II) and hexadecanoic acid (III). Of these, compound II displayed the most potent algicidal activity with IC₅₀ values of 4.9 and 14.1 µg/mL for H. akashiwo and P. micans, respectively. Compound I showed moderate algicidal activity with IC₅₀ values of 13.4 and 24.7 µg/mL for H. akashiwo and P. micans, respectively. These findings suggested that certain macroalgae or products therefrom could be used as effective biological control agents against red tide algae.     

Preparation of polysaccharides in different molecular weights from Ulva pertusa Kjellm （Chorophyta）

As molecular weight controls the biological activities of polysaccharides, screening the optimal molecular weight of polysaccharides is important in drug research and application. In this study, hydrogen peroxide was employed as oxidant, and temperature, reaction time, and concentration of polysaccharides and hydrogen peroxide were examined for their effects on the preparation of polysaccharides in different molecular weights from Ulva pertusa. Our experiment suggested that the optimal degradation concentrations for polysaccharides and hydrogen peroxide were 2.5% (w/v) and 8.0% (v/v), respectively. The range of degradation measured by relative viscosity was mainly controlled by temperature and time. Results revealed that 35℃ was the optimal temperature for obtaining low-degradation samples, and 50℃ was the most favorable temperature to accelerate the reaction to yield highly-degraded samples. Four samples in different molecular weights A, B, C and D were finally prepared. The controllability was evaluated by the relative standard deviation (RSD) of relative viscosity, and the peak molecular weights and the polydispersity indexes (Mw/Mn) of molecular weights were measured by high performance gel permeation chromatography (HPGPC).     

Steroids from green alga Chaetomorpha basiretorsa Setchell

Six steroids have been isolated from ethanolic extract of green alga Chaetomorpha basiretorsa Setehell by a combination of repeated normal phase silica gel and Sephadex LH-20 gel column chromatography as well as recrystallization. Using spectroscopic methods including MS and NMR, their structures were determined as β-lawsaritol (1), saringosterol (2), 24-hydroperoxy-24-vinyl-cholesterol (3), β-stigmasterol (4), stigmast-4-en-3α, 6β-diol (5), 29-hydroxystigmasta-5, 24 (28)-dien-3β-ol (6). All these compounds were obtained from this genus for the first time and they were inactive (IC5010 μg/ml) against KB, Bel-7402, PC-3M, Ketr 3 and MCF-7 cell lines.     

Acute toxicity of live and decomposing green alga Ulva (Enteromorpha) prolifera to abalone Haliotis discus hannai

From 2007 to 2009, large-scale blooms of green algae (the so-called “green tides”) occurred every summer in the Yellow Sea, China. In June 2008, huge amounts of floating green algae accumulated along the coast of Qingdao and led to mass mortality of cultured abalone and sea cucumber. However, the mechanism for the mass mortality of cultured animals remains undetermined. This study examined the toxic effects of Ulva (Enteromorpha) prolifera, the causative species of green tides in the Yellow Sea during the last three years. The acute toxicity of fresh culture medium and decomposing algal effluent of U. prolifera to the cultured abalone Haliotis discus hannai were tested. It was found that both fresh culture medium and decomposing algal effluent had toxic effects to abalone, and decomposing algal effluent was more toxic than fresh culture medium. The acute toxicity of decomposing algal effluent could be attributed to the ammonia and sulfide presented in the effluent, as well as the hypoxia caused by the decomposition process.     

Effect of UV-B irradiation on interspecific competition between Ulva pertusa and Grateloupia filicina

We report the effect of UV-B irradiation(9.6 kJ m-2day-1)on interspecific competition between two species of macroalgae,Ulva pertusa(U)and Grateloupia filicina(G),in co-culture.Growth of U.pertusa and G.filicina was inhibited by UV-B irradiation in mono-culture and specific growth rate (μ)declined as a result.Interspecific competition between U.pertusa and G.filicina was closely related to the initial weights when co-cultured.When initial ratios of U.pertusa(U)to G.filicina(G)were U:G=1.2:1 and 1:1,U.pertusa was the dominant algae.When the initial U:G ratio was 1:1.2,G.filicina was competitively dominant in the earlier stage,but U.pertusa grew faster,superseding G.filicina in the later stage.At initial ration U:G=1:1.4,G.filicina was predominant.Under UV-B irradiation,the competitive ability of G.filicina was weakened and the interspecific competitive balance favored U.pertusa,which suggests that G.filicina was more sensitive to UV-B irradiation.We also probed the potential allelopathic effects between the two species,which led to mutual growth inhibition.     

Effect of UV-B irradiation on interspecific competition between Ulva pertusa and Grateloupia filicina

We report the effect of UV-B irradiation (9.6 kJ m^?2 day^?1) on interspecific competition between two species of macroalgae, Ulva pertusa (U) and Grateloupia filicina (G), in co-culture. Growth of U. pertusa and G. filicina was inhibited by UV-B irradiation in mono-culture and specific growth rate (μ) declined as a result. Interspecific competition between U. pertusa and G. filicina was closely related to the initial weights when co-cultured. When initial ratios of U. pertusa (U) to G. filicina (G) were U:G=1.2:1 and 1:1, U. pertusa was the dominant algae. When the initial U:G ratio was 1:1.2, G. filicina was competitively dominant in the earlier stage, but U. pertusa grew faster, superseding G. filicina in the later stage. At initial ration U:G = 1:1.4, G. filicina was predominant. Under UV-B irradiation, the competitive ability of G. filicina was weakened and the interspecific competitive balance favored U. pertusa, which suggests that G. filicina was more sensitive to UV-B irradiation. We also probed the potential allelopathic effects between the two species, which led to mutual growth inhibition.     

Effects of dietary supplementation of Ulva pertusa and non-starch polysaccharide enzymes on gut microbiota of Siganus canaliculatus

Fishes represent the highest diversity of vertebrates; however, our understanding of the compositions and functions of their gut microbiota is limited. In this study, we provided the first insight into the gut microbiota of the herbivorous fish Siganus canaliculatus by using three molecular ecology techniques based on the 16 S r RNA genes(denaturing gradient gel electrophoresis, clone library construction, and highthroughput Illumina sequencing), and the Illumina sequencing technique is suggested here due to its higher overall coverage of the total 16 S r RNA genes. A core gut microbiota of 29 bacterial groups, covering 99.9% of the total bacterial community, was found to be dominated by Proteobacteria and Firmicutes in fish fed three dif ferent diets with/without the supplementation of U lva pertusa and non-starch polysaccharide(NSP) enzymes(cellulase, xylanase, and β-glucanase). Diverse potential NSP-degrading bacteria and probiotics(e.g., R uminococcus, Clostridium and Lachnospiraceae) were detected in the intestine of the fish fed U. pertusa, suggesting that these microorganisms likely participated in the degradation of NSPs derived from U. pertusa. This study supports our previous conclusion that U. pertusa-based diets are suitable for the production of S. canaliculatus with lower costs without compromising quality.     

The Ulva prolifera genome reveals the mechanism of green tides

The genome of green microalgae has rarely been reported.Ulva prolifera is a green microalga that has received much attention.Despite research articles about U.prolifera in recent years,we know very little about its genome.Therefore,the 87.9-Mb haploid genome(containing 10 311 protein-coding genes)of U.prolifera was studied,and the genome was compared with that of U.mutabilis,which is the only published Ulva species.Results showed that the two species are closely related.A phylogenetic tree was constructed among U.prolifera and other green algae available in GenBank,revealing the evolutionary status of U.prolifera in Chlorophyta.To understand why U.prolifer a could grow rapidly,we identified some genes related to growth,such as those involved in cell division,phosphorylation,and cell proliferation.In addition,genes related to stress resistance were found,which supports the notion that U.prolifera grows vigorously in nature.These results help to characterize green tides from a new perspective and reveal some important insight into the biology of U.prolifera.     

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 p H) 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), and they were equally important in impacting T. subcordiformis specific growth; the linear effect of p H was not significant( P 0.05); the interactive effect of temperature and p H was significant( P 0.05), whereas the temperature ′ salinity and salinity ′ p H interactions were not 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 R~2 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/p H 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.     

Morphology and molecular identification of Ulva forming green tides in Qingdao, China

Green tides are caused by the proliferation of chlorophytes under suitable hydrographic conditions.These blooms lead to environmental degradation and negatively impact the waters and seagrass beds,as well as fishing and other recreational activities in the bay.A comprehensive ecological understanding of the bloom dynamics,including the origin and persistence,is needed to foster management decisions.The algae in the great majority of green tide blooms usually belong to two genera of Ulvophyceae,Ulva and Enteromorpha.Ulva has been observed more often in recent years.In China,green tides occurred for the first time in the middle area of the Yellow Sea in 2007,and a large-scale algae blooming broke out in the middle and southern areas of the Yellow Sea in late May 2008.We identified them as Ulva prolifera by comparative analysis of the rDNA internal transcribed spacer 1 (ITS1),5.8S and ITS2 sequences in combination with microscopic observation.Morphological differences were found between the free-floating algae and the attached thalli.Various reproduction patterns of the free-floating algae include sexual,asexual and vegetative propagations,which played important roles in the long-term green tide persistence in China.The ITS sequences of the blooming algae were identical to those of the samples from the Lianyungang sea area but were different from the attached samples from the Qingdao sea area.The results infer that the blooms are originated from other sea areas rather than from the local attached populations.     

Extraction and PTP1B inhibitory activity of bromophenols from the marine red alga Symphyocladia latiuscula

Previously, we had characterized several structurally interesting brominated phenols from the marine red alga Symphyocladia latiuscula collected from various sites. However, Phytochemical investigations on this species collected from the Weihai coastline of Shandong Province remains blank. Therefore, we characterized the chemical constituents of individuals of this species collected from the region. Eight bromophenols were isolated and identified. Using detailed spectroscopic techniques and comparisons with published data, these compounds were identified as 2,3-dibromo-4,5-dihydroxybenzyl methyl ether (1), 3,5-dibromo-4-hydroxybenzoic acid (2), 2,3,6-tribromo-4,5-dihydroxymethylbenzene (3), 2,3,6-tribromo-4,5-dihydroxybenzaldehyde (4), 2,3,6-tribromo-4,5-dihydroxybenzyl methyl ether (5), bis(2,3,6-tribromo-4,5-dihydroxyphenyl)methane (6), 1,2-bis(2,3,6-tribromo-4,5-dihydroxyphenyl)-ethane (7), and 1-(2,3,6-tribromo-4,5-dihydroxybenzyl)-pyrrolidin-2-one (8). Among these compounds, 1 and 2 were isolated for the first time from S. latiuscula. Each compound was evaluated on the ability to inhibit protein tyrosine phosphatase 1B (PTP1B), which is a potential therapeutic target in the treatment of type 2 diabetes. Bromophenols 5, 6, and 7 showed strong activities with IC50 values of 3.9, 4.3, and 3.5 μmol/L, respectively. This study provides further evidence that bromophenols are predominant among the chemical constituents of Symphyocladia, and that some of these compounds may be candidates for the development of anti-diabetes drugs.     

ARTIFICIAL PROPAGATION AND BREEDING OF MARINE FISH IN CHINA

Since the 1990s, artificial propagation and breeding technique of marine fish in China have developed by way of increasing species and fry numbers, with special stress laid on valuable species. Large quantities of artificial fry can meet the needs of both marine cage culture and pond culture for most species. Experimental results obtained by scientists have been put into use in actual production.Fish fry production has entered a period of sustainable development. So far, at least 44 species (21 families) of marine fish have been successfully bred in China. The artificial fry number of large yellow croaker ( Pseudosciaena crocea) exceeded 300 million in 1999. The species whose artificial fry numbers have each surpassed 10 million annually are red drum ( Sciaenops ocellatus ), Japanese seabass ( Lateolabrax japonicus), cuneate drum (Nibea miichthioides ), spring spawning red seabream (Pagrosomus major ) and threebanded sweethp ( Plectorhynchus cinctus ). Millions of artificial fry are bred annually in the species of black porgy ( Sparus macrocephalus ), Russell‘ s snapper ( Lutjanus russeUi ), javelin gnmt ( Pomadasys hasta ), miiuy croaker ( Miichthys miiuy) and skewband gnmt ( Hapalogenys nitens ). The fish in the family Sciaenidae are the main species in artificial propagation and breeding. Some problems and prospects on marine fish culture and stock enhancement are also discussed and some proposals for sustainable development are put forward in this article.     

Steroids from green alga <Emphasis Type="Italic">Chaetomorpha basiretorsa</Emphasis> Setchell

Six steroids have been isolated from ethanolic extract of green alga Chaetomorpha basiretorsa Setchell by a combination of repeated normal phase silica gel and Sephadex LH-20 gel column chromatography as well as recrystallization. Using spectroscopic methods including MS and NMR, their structures were determined as β-1awsaritol （1）, saringosterol （2）, 24-hydroperoxy-24-vinyl- cholesterol （3）, β-stigmasterol （4）, stigmast-4-en-3α, 6β-diol （5）, 29-hydroxystigmasta-5, 24 （28）-dien-3β-ol （6）. All these compounds were obtained from this genus for the first time and they were inactive （IC50〉10μg/ml） against KB, Bel-7402, PC-3M, Ketr 3 and MCF-7 cell lines.     

Flocculation of Ulva microscopic propagules using modified clay: a mesocosm experiment

Microscopic propagules of Ulva species(UMPs) spread on Subei Shoal are believed to play a significant role in the formation of the Yellow Sea green tide.Previous laboratory and ship-based studies indicated that modified clay(MC) can effectively remove UMPs from the water column and restrict their germination.To evaluate the effectiveness of this method under natural conditions,a mesocosm experiment was conducted on the Zhugensha sandbank on Subei Shoal.The results show that the suspended particles on the sandbank would partially disturb the flocculation process between the MC and UMPs.However,the MC at a proper dose could effectively remove UMPs from the water column and decrease the adhesion and germination of UMPs on the nylon ropes that comprised the mariculture rafts by75%.This method was proven a potential strategy to restrict the initial biomass accumulation of green algae on Subei Shoal.However,field trials in larger time-space scales are still needed to assess the efficiency of this method when used in the open sea.     

Electron microscope evidence of virus infection in cultured marine fish

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Immunological cross-reactions between P700 chlorophyll-proteins isolated from two marine green algae and one higher plant

P₇₀₀ Chl-protein was isolated from a marine green algaBryopsis corticulans with SDS-resolved thylakoid membranes by SDS-PAGE. After elution from the gel, the recovered protein revealed a 100 KD polypeptide by re-electrophoresis. The same SDS-PAGE procedure was used to isolate P₇₀₀ Chl-proteins from spinach (Spinacea oleracea) and another marine green algaCodium fragile. Polyclonal antibodies toBryopsis P₇₀₀ protein were raised in rabbits. The antibodies were shown to cross-react with P₇₀₀ Chl-protein and Chl-protein complexes containing P₇₀₀ protein fromB. Corticulans, C. f ragile, and even spinach. Results indicate similarity of the amino acid sequences of the P₇₀₀ Chl-proteins and the highly conserved structure of the apoproteins of phylogenetically distant species over evolution. The antibodies cross-react with none of the components of PSII in the species tested, indicating an independent pathway of evolution of photosystem I and photosystem II of later origin. Contribution No. 1717 from the Institute of Oceanology, Academia Sinica     

Tempo-spatial distribution of Ulva spp. micro-propagules in the Yellow Sea during and after green tide in 2019

Journal of Oceanology and Limnology - Large-scale green tides occur frequently in summer in the Yellow Sea in recent decade. Micro-propagules, providing “seeds” for the seaweeds, play...