Isolation of Chloroplasts from Marine Microalga Isochrysis galbana Parke for Their Lipid Composition Analysis

Marine microalga Isochrysis galbana is an important feed species with a high nutritional value.Different from other uni-cellular algae,its cell contains two chloroplasts which are the major sites for lipid synthesis.Here,we optimized a chloroplast isola-tion approach suitable for the isolation of I.galbana chloroplasts and determined the purity and integrity of the isolated chloroplasts through microscopic observations and enzyme activity assay.The chloroplast lipids were analyzed with a ultrahigh-performance li-quid chromatography-Q Exactive Orbitrap-mass spectrometry.This newly developed isolation approach is simple and reliable to isolate chloroplasts with high integrity and purity.The average yield of intact chloroplasts was 15.3%±0.1%.Glycolipids and acyl-glycerols were the main chloroplast lipids.Glycolipids accounted for 56.6%of chloroplast lipid.Digalactosyldiacylglycerol(DGDG),monogalactosyldiacylglycerol(MGDG)and sulfoquinovosyldiacylglycerol(SQDG)were the main glyceroglycolipids.The fatty acyl R1/R2 were mostly 18:4/16:1,18:3/16:1 and 18:4/18:5 in DGDGs,14:0/18:4,18:4/18:5,18:4/18:4 and 18:3/18:4 in MGDGs and 16:0/14:0,16:0/18:3,and 18:4/18:3 in SQDGs.In addition,diacylglycerol(DAG)was the most abundant acylglycerols;the content of 22:6/18:4-DAG was the highest.There was a little amount of glycosphingolipid(GSL)in chloroplast.Digalactosylmonoglyceride(DGMG),monogalactosylmonoglyceride(MGMG),sulfoquinovosylmonoacylglycerol(SQMG),monoglyceride(MAG),phospholi-pids(PLs),ceramide(Cer)and betaine lipids were nearly undetectable in chloroplast.The fatty acid proportions of DGDGs,MGDGs,SQDGs,DAGs,triglycerides(TAGs)and GSLs were either higher or lower than or similar to those of whole-cell.Collectively,our isolation approach is applicable to many aspects of chloroplast biology,and may offer a reference for the isolation of chloroplasts from other marine microalgae.     

Removal of phenol by Isochrysis galbana in seawater under varying temperature and light intensity

Phenol is a common industrial chemical produced and transported worldwide largely. Therefore, accidental spillage of phenol in the ocean causes an increasing concern. Microalgae are promising to remove phenol from marine waters. However, temperature and light intensity are two main factors that markedly influence biodegradation in marine environments. In this study, a marine golden alga Isochrysis galbana is selected to research the removal of phenol under different temperatures (10–30°C) and light intensities (0–240 µmol/(m2·s)). The results show that the most suitable temperature and light intensity for phenol removal are 20°C and 180 µmol/(m2·s), respectively, and 100 mg/L of phenol can be completely removed by microalga in 24 h at these conditions. I. galbana can also remove phenol under dark and low-temperature conditions. The removal of phenol by I. galbana at diverse temperatures and light intensities conform to first-order kinetics, and the process under dark conditions conform to zero-order kinetics. Thus, I. galbana can be used in the in-situ bioremediation of polluted seawater by phenol.     

Effect of Fe3+ on the growth and lipid content of Isochrysis galbana

Inducing lipid accumulation in microalgae cells without suppressing cell growth is vital to the economical production of biodiesel from microalgae. In two experiments, we demonstrate that the cell concentration and lipid content of marine microalgae Isochrysis galbana depend upon the iron concentration in the growth media. In Experiment I, adding chelated FeCl 3 to the medium at the late exponential growth phase prolonged this phase and increased the lipid content in I. galbana cells. The fi nal cell density and lipid content of I. galbana supplemented with chelated FeCl 3 was approximately 2 and 1.65 times higher than that of non-supplemented cultures, respectively. In Experiment II, I. galbana cells in the late exponential phase were collected and re-inoculated into new media containing Fe 3+ at various concentrations. The fi nal cell concentration and lipid content were maximized at the highest iron concentration(38% biomass by dry weight at 1.2×10-5 mol/L Fe 3+). In this study, intracellular neutral lipid storage was evaluated by fl uorescent spectrophotometry using fl uorochrome Nile red, and the measurement conditions were optimized.     

Isolation and characterization of a fucoidan-degrading bacterium from Laminaria japonica

Fucoidan, a polysaccharide containing abundant fucose and sulfate ester group, was prepared from Laminaria japonica. In order to obtain fucoidan-degrading enzyme, bacteria capable of degrading fucoidan were screened from kelp. A bacterial strain named RC2-3 was obtained, which degraded fucoidan by the maximum extent of 54% ± 1.3%, the highest among all bacterial isolates. High-performance size exclusion chromatography(HPSEC) showed that the molecular weight of fucoidan was gradually reduced by RC2-3 with culturing time, suggesting the production of fucoidan-degrading enzyme by RC2-3. Phylogenetic analysis of partial 16S ribosomal RNA gene(16S rDNA) sequence showed that RC2-3 belonged to the family Flavobacteriaceae. However, it showed different physiological and biochemical characteristics from the known Flavobacteriaceae members producing fucoidan-degrading enzyme, thus RC2-3 was proposed to be a new member of this family.     

Isolation and characterization of phosphate-solubilizing bacteria from seagrass rhizosphere soil

Phosphate-solubilizing bacterial strains(6 Nos.) were isolated from the rhizosphere soils of two seagrasses(Halophila ovalis(R.Br.) Hook and Halodule pinifolia(Miki) Hartog) in the Vellar estuary.Experimental studies found that the strain PSSG6 was effective in phosphate solubilization with Phosphate Solubilization efficiency index E = 375 ± 8.54,followed by the strain PSSG5 with Phosphate Solubilization efficiency index E = 275 ± 27.3.Of the 6 strains isolated,the strains PSSG4 and PSSG5 be-longed to the genus Bacillus,and PSSG1,PSSG2 and PSSG3 were identified as Citrobacter sp.,Shigella sp.,and Klebsiella sp.,respectively,by conventional method,and PSSG6 was identified as Bacillus circulans using conventional and molecular methods.     

Isolation and characterization of AHL-degrading bacteria from fish and pond sediment

Quorum sensing(QS)disruption is considered as a potential alternative strategy to combat bacterial diseases in aquaculture.In this study,we isolated and identified bacteria degrading QS molecules from pond sediment and fish intestine.A total of 132 strains were obtained in the enrichment culture,of which two strains were identified as Enterobacter sp.f003 and Staphylococcus sp.sw120,being isolated from the fish intestine and pond sediment,respectively.We found that strains f003 and sw120 could degrade acyl-homoserine lactones(AHLs)and cause no hemolysis of sheep red blood cells.The AHL lactonase(aiiA)homologous gene in the two strains was detected in PCR amplification and the high-degrading activity to N-hexanoyl-L-homoserine lactone(C6-HSL)and AHLs secreted from pathogenic Aeromonas hydrophila was assessed.Meanwhile,the artificial infection of cyprinid Carassius auratus gibelio with intraperitoneal injection showed that the two strains were avirulent.Therefore,the obtained indigenous bacteria are candidate probiotics against pathogenic A.hydrophila in aquaculture.     

Isolation and characterization of venom from nematocysts of jellyfish Rhopilema esculentum Kishinouye

The present work is the first report of the biochemical characterization of the venom from nematocysts of the jellyfish Rhopilema esculentum Kishinouye. The nematocysts were isolated by autolysis and centrifugation and separated by flow cytometry. Four types of nematocysts were identified: mastigophores, euryteles, and atrichous and holotrichous isorhiza. SDS-PAGE and amino acid analyses demonstrated that most of the proteins in the nematocyst extract were between 10 kDa and 40 kDa, and that glutamic acid was the main amino acid. A hemolytic activity assay showed that the activity of the nematocyst venom (RNV) was strongest in Tris-HCl buffer (50 mmol/L, pH 7.8, 5% glycerol, 0.5 mmol/L EDTA, 0.1 mol/L NaCl). The hemolytic activity was related to protein concentration and the HU50 against chicken erythrocytes was 0.91 μg/mL.     

Isolation and characterization of collagen from squid (Ommastrephes bartrami) skin

Collagen of squid (Ommastrephes bartrami) skin was examined in the present study. Histology showed that collagen fiber in the skin was partially cross-linked with muscle fiber. Acid-solubilized collagen (ASC) and pepsin-solubilized collagen (PSC) were extracted from the skin and characterized. The results of amino acid composition and electrophoretic patterns revealed that ASC and PSC were both type I collagen, containing α1 and α2 chains. FTIR (fourier transform infrared spectroscopy) investigations confirmed the existence of helical arrangements in PSC of squid skin. The denaturation temperature (T_d) and shrinkage temperature (T_s) of PSC were 29.4°C and 52.8°C, respectively.     

Construction and characterization of Gal-chitosan graft methoxy poly (ethylene glycol) (Gal-CS-mPEG) nanoparticles as efficient gene carrier

In the present study, galactosylated chitosan (Gal-CS) was conjugated with methoxy poly(ethylene glycol) (mPEG) as a hydrophilic group. The structure of Gal-CS-mPEG polymer was characterized and the nanoparticles (NPs) were prepared using ironic gelation method. The study was designed to investigate the characteristics and functions of Gal-CS-mPEG NPs. The morphology of Gal-CS-mPEG NPs was observed by SEM and it was a compact and spherical shape. The size of the NPs was approximately 200 nm in diameter under the ideal process parameters. The interaction between Gal-CS-mPEG NPs and pDNA, and the protection of pDNA against DNase I and serum degradation by Gal-CS-mPEG NPs were evaluated. Agarose gel electrophoresis results showed that Gal-CS-mPEG NPs had strong interaction with pDNA at the weight ratio of 12:1, 4:1 and 2:1 and could protect pDNA from DNase I and serum degradation. Gal-CS-mPEG NPs exhibited high loading efficiency and sustainable in vitro release. The blood compatibility studies demonstrated that Gal-CS-mPEG NPs had superior compatibility with erythrocytes in terms of aggregation degree and hemolysis level. Gal-CS-mPEG NPs showed no cytotoxicity on L929 cells, which is a normal mouse connective tissue fibroblast, but showed inhibitory effects on the proliferation of Bel-7402 cells, which is a liver cancer cell line. In conclusion, Gal-CS-mPEG NP is a bio-safe and efficient gene carrier with potential application in gene delivery.     

Isolation and characterization of fucoidans from five brown algae and evaluation of their antioxidant activity

In this study, we evaluated the chemical property and antioxidant activity of fucoidans isolated from brown algae, Laminaria japonica(LJF), Lessonia nigrescens(LNF), Lessonia trabeculata(LTF), Ascophyllum mackaii(AMF), and Ecklonia maxima(EMF). LJF was less in sulfate content(14.16%) and more in galactose and mannose content(1.08 and 0.68) than the documented early. EMF contained 20%–30% of sulfate and fucose, 0.97 in molar ratio which was lower than that of sulfate to other four fucoidans(1.21–1.41). AMF(162 kDa) and EMF(150 kDa) were the first two largest in molecular weight, which were followed by LJP(126 kDa), LNF(113 kDa) and LTF(105 kDa). The fucoidans isolated these algae showed a wide range of antioxidant activity in vitro. It was found that the reducing power of the isolated fucoidans was positively correlated with their sulfate content and molecular weight. In addition, LNF and LTF at low concentrations exhibited high superoxide and hydroxyl radical scavenging activity. This demonstrated that low molecular weight fucoidans may perform a high antioxidant activity.     

Isolation and characterization of a marine bacterium producing protease from Chukchi Sea, Arctic

A Gram negative bacterium Ar/W/b/75°25＇N/1 producing extracellular alkaline protease was isolated from surface water of latitude 75°25＇N, and longitude 162°25＇W in Chukchi sea, Arctic. The strain can grow at the temperature range from 7℃ to 30℃, and grow better at 30(℃. It can not grow at 40℃. Keeping certain salinity concentration in medium is necessary for cell growth. It grows well in medium containing salinity concentration from 0. 5 % to 10 % sodium chloride. Glucose, sucrose and soluble starch can be utilized by the strain, among which glucose is the optimal carbon source. Peptone is the optimal organic nitrogen source for cell growth and protease producing, and ammonium nitrate is the optimal inorganic nitrogen source.About 75.7% of total protease of the strain are extracellular enzyme. Optimal temperature for proteolytic activity is at 40℃. Protease of the strain keeps stable below 40℃, and shows high proteolytic activity within the pH range from 7 to 11.     

Isolation and characterization of bioactive fungi from shark Carcharodon carcharias' gill with biopharmaceutical prospects

Until recently,little was known about the fungi found in shark gills and their biomedicinal potential. In this article,we described the isolation,bioactivity,diversity,and secondary metabolites of bioactive fungi from the gill of a shark(Carcharodon carcharias). A total of 115 isolates were obtained and grown in 12 culture media. Fifty-eight of these isolates demonstrated significant activity in four antimicrobial,pesticidal,and cytotoxic bioassay models. Four randomly selected bioactive isolates inhibited human cancer cell proliferation during re-screening. These active isolates were segregated into 6 genera using the internal transcribed spacer-large subunit(ITS-LSU) rDNA-sequence BLAST comparison. Four genera,Penicillium,Aspergillus,Mucor,and Chaetomium were the dominant taxa. A phylogenic tree illustrated their intergenera and intragenera genetic diversity. HPLC-DAD-HRMS analysis and subsequent database searching revealed that nine representative strains produced diverse bioactive compound profiles. These results detail the broad range of bioactive fungi found in a shark's gills,revealing their biopharmaceutical potential. To the best of our knowledge,this is the first study characterizing shark gill fungi and their bioactivity.     

Cloning and characterization of a delta-6 desaturase encoding gene from Nannochloropsis oculata

A gene(NANOC-D6D) encoding a desaturase that removes two hydrogen atoms from fatty acids at delta 6 position was isolated from a cDNA library of Nannochloropsis oculata(Droop) D.J.Hibberd(Eustigmatophyceae).The unicellular marine microalga N.oculata synthesizes rich long chain polyunsaturated fatty acids(LCPUFAs),including eicosapentaenoic acid(20:5n-3,EPA).The deduced protein contains 474 amino acids that fold into 4 trans-membrane domains.The neighbor-joining phylogenetic tree indicates that NANOC-D6D is ...     

Isolation and characterization of halophilic bacteria and archaea from salt ponds in Hangu Saltworks,Tianjin, China

A total of 26 isolates were obtained from solar salt ponds of different salinities(100, 150, 200, and 250) in Hangu Saltworks Co. Ltd., Tianjin, China. Phylogenetic analysis of 16 SrRNA gene sequences indicated that five bacteria genera H alomonas, Salinicoccus, Oceanobacillus, Gracibacillus, and Salimicrobium and one archaea genera H alorubrum were present. The genus H alomonas was predominant with eight strains distributed in a salinity range of 100–200, followed by H alorubrum with six strains in salinity 250. Based on the genus and original sampling salinity, eight bacterial and two archaeal isolates were selected for further morphological, physiological, and biochemical characterization. All of the bacterial strains were moderately halophilic with the optimal salinity for growth being either 50 or 100, while two archaeal strains were extremely halophilic with an optimal growth salinity of 200. Additionally, we put forth strain SM.200-5 as a new candidate S alimicrobium species based on the phylogenic analysis of the 16 SrRNA gene sequence and its biochemical characteristics when compared with known related species.     

Purification and characterization of an alkaline protease from Acetes chinensis

An alkaline protease from Acetes chinensis was purified and characterized in this study. The steps of purification include ammonium sulfate precipitation, ion-exchange chromatography with Q-sepharose Fast Flow, gel filtration chromatography with S300 and the second ion-exchange chromatography with Q-sepharose Fast Flow. The protease was isolated and purified, which was present and active on protein substrates (azocasein and casein). The specific protease activity was 17.15 folds and the recovery was 4.67. The molecular weight of the protease was estimated at 23.2 kD by SDS-PAGE. With azocasein as the susbstrate, the optimal temperature was 55°C and the optimal pH value was 5.5. Ion Ca²⁺ could enhance the proteolytic activity of the protease, while Cu²⁺, EDTA and PMSF could inhibit its activity.     

Characterization of defensin gene from abalone Haliotis discus hannai and its deduced protein

Defensin is one of preserved ancient host defensive materials formed in biological evolution. As a regulator and effector molecule, it is very important in animals' acquired immune system. This paper reports the defensin gene from the mixed liver and kidney cDNA library of abalone Haliotis discus hannai Ino. Sequence analysis shows that the gene sequence of full-length cDNA encodes 42 mature peptides (including six Cys), molecular weight of 4 323 Da, and pI of 8.02. Amino acid sequence homology analysis shows that the peptides are highly similar (70% in common) to other insects defensin. Because of a typical insect-defensin structural character of mature peptide in the secondary structure, the polypeptide named Haliotis discus defensin (hd-def), a novel of antimicrobial peptides, belongs to insects defensin subfamily. The RT-PCR result of Haliotis discus defensin shows that the gene can be expressed only in the hepatopancreas by Gram-negative and positive bacteria stimulation, which is ascribed to inducible expression. Therefore, it is revealed that the Haliotis discus defensin gene expression was related to the antibacterial infection of Haliotis discus hannai Ino.