Studies on Bioflocculant Production by Pseudoalteromonas sp.NUM8, a Marine Bacteria Isolated from the Circulating Seawater

A bioflocculant producing potential bacteria was isolated from the circulating seawater of bio-filter using streak plate methods. The bacteria was identified through biochemical characteristics, partial 16 S ribosomal ribonucleic acids(rRNA), nucleotide sequencing, and BLAST analysis of the gene sequence that showed the bacteria have 99% similarity to Pseudoalteromonas sp.and deposited in GenBank as Pseudoalteromonas sp. NUM8 with accession number JX435820. Influences of time course assay,carbon sources, nitrogen sources, inoculum size, as well as initial pH on the bacteria producing extracellular bioflocculant activity were investigated. The results showed that the strain optimal production period of microbial bioflocculant was at 72 h(flocculating activity of 94.5%), then dropped slowly. The bacteria optimally produced the bioflocculant when 1.0% sucrose and 0.5% sodium nitrate were used as sole sources of carbon and nitrogen with flocculating activities of 92.8% and 93.8% respectively. Also, the bacteria produced the bioflocculant optimally when initial pH of the medium was 5.0(flocculating activity 93.2%), and when Ca~(2+)was used as cation(flocculating activity 93.4%). The culture condition of inoculum size of 3%(v/v) was optimal flocculant production(flocculating activity 94.4%). Composition analyses indicated the bioflocculant to be principally a glycoprotein made up of about 34.3% protein and 63.4% total carbohydrate.     

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.     

Purification and characterization of 2-haloacid dehalogenase from marine bacterium Paracoccus sp. DEH99, isolated from marine sponge Hymeniacidon perlevis

2-haloacid dehalogenases constitute a group of dehalogenases which are capable of dehalogenating the halogenated organic compounds. So far, the 2-haloacid dehalogenases have been found in many bacteria, but not in Paracoccus genus. In the present study, one enzyme 2-haloacid dehalogenase(designated as Deh99), induced by DL-2-chloropropionate(DL-2-CPA), was purified from the marine bacterium Paracoccus sp. DEH99, isolated from marine sponge Hymeniacidon perlevis. The enzyme of Deh99 was purified to homogeneity by ammonium sulfate precipitation, ion exchange chromatography(Q-Sepharose HP), and Superdex 200 gel filtration chromatography. The molecular weight of Deh99 was estimated to be 25.0 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE), and 50.0 kDa natively by gel filtration chromatography. The enzyme of Deh99 stereospecifically dehalogenated L-2-CPA to produce D-lactate, with an apparent Michaelis-Menten constant(Km) value of 0.21 mmol L-1 for L-2-CPA. The optimal pH and temperature for Deh99 activity were 10.0 and 40℃, respectively. The enzyme of Deh99 acted on short-carbon-chain 2-haloacids, with the highest activity towards monochloroacetate. The activity of Deh99 was slightly affected by DTT and EDTA, but strongly inhibited by Cu2+ and Zn2+. The enzyme of Deh99 shows unique substrate specificity and inhibitor sensitivities compared to previously characterized 2-haloacid dehalogenases and is the reported one about purified 2-haloacid dehalogenase isolated from the bacteria of Paracoccus genus.     

Inhibitory activity of an extract from a marine bacterium Halomonas sp. HSB07 against the red-tide microalga Gymnodinium sp. （Pyrrophyta）

In recent years, red tides occurred frequently in coastal areas worldwide. Various methods based on the use of clay, copper sulfate, and bacteria have been successful in controlling red tides to some extent. As a new defensive agent, marine microorganisms are important sources of compounds with potent inhibitory bioactivities against red-tide microalgae, such as Gymnodinium sp. （Pyrrophyta）. In this study, we isolated a marine bacterium, HSB07, from seawater collected from Hongsha Bay, Sanya, South China Sea. Based on its 16S rRNA gene sequence and biochemical characteristics, the isolated strain HSB07 was identified as a member of the genus Halomonas. A crude ethyl acetate extract of strain HSB07 showed moderate inhibition activity against Gymnodinium sp. in a bioactive prescreening experiment. The extract was further separated into fractions A, B, and C by silica gel column chromatography. Fractions B and C showed strong inhibition activities against Gymnodinium. This is the first report of inhibitory activity of secondary metabolites of a Halomonas bacterium against a red-tide-causing microalga.     

Cloning,Expression and Characterization of a Lipase Gene from Marine Bacterium Pseudoalteromonas lipolytica SCSIO 04301

Absract A lipase gene, lip1233, isolated from Pseudoalteromonas lipolytica SCSIO 04301, was cloned and expressed in E. coli. The enzyme comprised 810 amino acid residues with a deduced molecular weight of 80 k Da. Lip1233 was grouped into the lipase family X because it contained a highly conserved motif GHSLG. The recombinant enzyme was purified with Ni-NTA affinity chromatography. The optimal temperature and p H value of Lip1233 were 45℃ and 8.0, respectively. It retained more than 70% of original activity after being incubated in p H ranging from 6.0 to 9.5 for 30 min. It was stable when the temperature was below 45℃, but was unstable when the temperature was above 55℃. Most metal ions tested had no significant effect on the activity of Lip1233. Lip1233 remained more than original activity in some organic solvents at the concentration of 30%(v/v). It retained more than 30% activity after incubated in pure organic solvents for 12 h, while in hexane the activity was nearly 100%. Additionally, Lip1233 exhibited typical halotolerant characteristic as it was active under 4M Na Cl. Lip1233 powder could catalyze efficiently the synthesis of fructose esters in hexane at 40℃. These characteristics demonstrated that Lip1233 is applicable to elaborate food processing and organic synthesis.     

Cloning,expression and characterization of a lipase gene from marine bacterium <Emphasis Type="Italic">Pseudoalteromonas lipolytica</Emphasis> SCSIO 04301

A lipase gene, lip1233, isolated from Pseudoalteromonas lipolytica SCSIO 04301, was cloned and expressed in E. coli. The enzyme comprised 810 amino acid residues with a deduced molecular weight of 80 kDa. Lip1233 was grouped into the lipase family X because it contained a highly conserved motif GHSLG. The recombinant enzyme was purified with Ni-NTA affinity chromatography. The optimal temperature and pH value of Lip1233 were 45°C and 8.0, respectively. It retained more than 70% of original activity after being incubated in pH ranging from 6.0 to 9.5 for 30 min. It was stable when the temperature was below 45°C, but was unstable when the temperature was above 55°C. Most metal ions tested had no significant effect on the activity of Lip1233. Lip1233 remained more than original activity in some organic solvents at the concentration of 30% (v/v). It retained more than 30% activity after incubated in pure organic solvents for 12 h, while in hexane the activity was nearly 100%. Additionally, Lip1233 exhibited typical halotolerant characteristic as it was active under 4M NaCl. Lip1233 powder could catalyze efficiently the synthesis of fructose esters in hexane at 40°C. These characteristics demonstrated that Lip1233 is applicable to elaborate food processing and organic synthesis.     

Optimization of culturing condition and medium composition for the production of alginate lyase by a marine <Emphasis Type="Italic">Vibrio</Emphasis> sp. YKW-34

Carbohydrases secreted by marine Vibrio sp. YKW-34 with strong Laminaria cell wall degrading ability were screened, and among them alginate lyase was found to be dominant. The effects of medium composition and culturing condition on the production of alginate lyase by marine Vibrio sp. YKW-34 in flask were investigated in this study. In the culture medium of marine broth, no alginate lyase was produced. The activity of the alginate lyase, after being induced, reached 5 UmL^-1. The best inoculum volume and inoculum age were 10% and 12h, respectively. The optimal temperature for alginate lyase production was 25℃. The fermentation medium was composed of 0.5% of Laminaria powder and 0.2% of KNO3 with an initial acidity of pH 8.0. Alginate could induce alginate lyase production but not as efficiently as Laminaria powder did. The addition of fucoidan, cellulose and glucose had negative effect on the alginate lyase production. Other kinds of nitrogen sources, such as yeast extract, beef extract and peptone, had positive effect on the growth of the microorganism and negative effect on alginate lyase production. In addition, the time course of alginate lyase production under the optimized condition was described. The optimal harvest time was 48 h.     

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.     

Characteristics of intracellular polyphosphate granules and phosphorus-absorption of a marine polyphosphate-accumulating bacterium,Halomonas sp. YSR-3

Journal of Oceanology and Limnology - Halomonas sp. YSR-3 was isolated from the Yellow Sea and identified as a polyphosphate-accumulating bacterium and the characteristics of its intracellular...     

Cold-adaptive alkaline protease from the psychrophilic Planomicrobium sp.547: enzyme characterization and gene cloning

A psychrophilic bacterium strain 547 producing cold-adaptive alkaline protease was isolated from the deep sea sediment of Prydz Bay, Antarctica. The organism was identified as a Planomicrobium species by 16S rRNA analysis. The optimal and highest growth temperatures for strain 547 were 15℃ and 30℃, respectively. The extracellular protease was purified by ammonium sulfate precipitation and DEAE cellulose-52 chromatography. The optimal temperature and pH for the activity of the purified enzyme were 35 ℃ and pH 9.0, respectively. The enzyme retained approximately 40% of its activity after 2 h of incubation at 50℃. The enzymatic activity was inhibited by 1 mmol/L phenylmethyl sulfonylfluoride (PMSF) and hydrochloride 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF), indicating that it was a serine protease. The presence of Ca2+ and Mn2+ increased the activity of the enzyme. The protease gene with a size of 1 269 bp was cloned from Planomicrobium sp. 547 using primers designed based on the conserved sequences of proteases in GenBank. The Planomicrobium sp. 547 protease contained a domain belonging to the peptidase S8 family, which has a length of 309 amino acid (AA) residues. The alignment and phylogenetic analysis of the AA sequence indicated that the protease belonged to the subtilisin family.     

Optimization of culture conditions and medium composition for the marine algicidal bacterium Alteromonas sp. DH46 by uniform design

Harmful algal blooms (HABs) have led to extensive ecological and environmental issues and huge economic losses. Various HAB control techniques have been developed, and biological methods have been paid more attention. Algicidal bacteria is a general designation for bacteria which inhibit algal growth in a direct or indirect manner, and kill or damage the algal cells. A metabolite which is strongly toxic to the dinoflagellate Alexandrium tamarense was produced by strain DH46 of the alga-lysing bacterium Alteromonas sp. The culture conditions were optimized using a single-factor test method. Factors including carbon source, nitrogen source, temperature, initial pH value, rotational speed and salinity were studied. The results showed that the cultivation of the bacteria at 28°C and 180 r min^?1 with initial pH 7 and 30 salt contcentration favored both the cell growth and the lysing effect of strain DH46. The optimal medium composition for strain DH46 was determined by means of uniform design experimentation, and the most important components influencing the cell density were tryptone, yeast extract, soluble starch, NaNO₃ and MgSO₄. When the following culture medium was used (tryptone 14.0g, yeast extract 1.63g, soluble starch 5.0 g, NaNO₃ 1.6 g, MgSO₄ 2.3 g in 1L), the largest bacterial dry weight (7.36 g L^?1) was obtained, which was an enhancement of 107% compared to the initial medium; and the algal lysis rate was as high as 98.4% which increased nearly 10% after optimization.     

Identification of a Long-Chain Fatty Acid Elongase from Nannochloropsis sp. Involved in the Biosynthesis of Fatty Acids by Heterologous Expression in Saccharomyces cerevisiae

The marine microalga Nannochloropsis sp. contains various elongases and desaturases that are critical for biosynthesis of polyunsaturated fatty acids. A full-length cDNA encoding a long-chain fatty acid elongase, named Ns FAE, was cloned from Nannochloropsis sp.. The open reading frame of Ns FAE(GenBank accession no. MF680548) consisted of 1068 bp and encoded a predicted protein of 355 amino acids with molecular mass 38.8 k Da. The deduced polypeptide showed 43%–44% identity to fatty acyl elongases from other algae. RT-PCR experiments indicated that the Ns FAE gene exhibited the highest expression in Nannochloropsis sp. at 72 h(i.e., during the third growth stage) and the expression was significantly lower in the other four growth stages. Plasmid p Ns FAE-CRISPR and a recombinant DNA fragment(ADH1p-Ns FAE-CYCt) were transformed into Saccharomyces cerevisiae strain BY4742 using the CRISPR-Cas system. Yeast transformants containing Ns FAE produced three fatty acids not normally present in wild-type BY4742-linoleic acid, linolenic acid and eicosadienoic acid-indicating that Ns FAE encodes a functional elongase enzyme.     

Study on changes of plasmalemma permeability and some primary inorganic ions of Antarctic ice microalgae （Chlamydomonas sp. ICE-L） in the low-temperature stress

The changes of plasmalemma permeability and some primary inorganic ions of Antarctic ice microalgae （ Chlamydomonas sp. ICE-L） in the low-temperature stress were examined. The plasmalemma of 1CE-L could maintain the stability at the freezing condition of -6 ℃. That signifies that it could maintain the proper function of plasmalemma and stability of the intracellular environment during sea ice formation. The function of inorganic ions on low-temperature adaptation of ICE-L was investigated by using the X-ray microanalysis method. Low temperature （0 - -6 ℃ ） induces Ca^2 ＋ concentration increment of cytoplasm, but after 24 h the content decrease quickly to normal value. As a matter of fact, Ca^2 ＋ plays an important role as the second messenger in the low temperature adaptation of ICE-L. In addition, low temperature also influences on the other primary inorganic ions transfer and the cell maintains activity by keeping ratio balance among different ions. Above all, it is necessary for Antarctic ice microalgae to survive and breed by maintaining the stability of K^ ＋ content and the balance of Na^ ＋/Cl^ -.     

Morphology and phylogeny of Chrysogorgia pinniformis sp.nov.and C.varians sp.nov.,two golden corals from the Caroline seamounts in the tropical Western Pacific Ocean

Two new species of Chrysogorgia Duchassaing Michelotti,1864 collected from the Caroline seamounts in the tropical Western Pacific Ocean are described.Chrysogorgia pinniformis sp.nov.belongs to Versluys' group C(Squamosae typicae) with only scales in polyp body wall and tentacles.C.pinniformis sp.nov.is characterized by large branches pinnately branched and forming multiple fans with its small branches regularly and quasi-dichotomous branched,and scales and rods present in the polyp mouth area.It is most similar to C.pinnata Cairns,2007 by the pinnate trait,but differs from the latter by its group C pattern(vs.group A,Spiculosae) and having sclerites present in the polyp mouth area(vs.absent).Chrysogorgia varians sp.nov.belongs to the Chrysogorgia group A(Spiculosae) with rods distributed in the polyp body wall and tentacles.It is characterized by warty rods and elongated scales in the tentacles,many warts and ridges on the scales,conspicuously toothed margins at the rounded ends in the pinnules,and small rods with ridge-like warts in the polyp mouth area.This species was frequent and abundant in the Caroline seamounts during our cruises and its morphological variability in growth period was obvious.The phylogenetic analyses based on mtMutS and 28 S rDNA regions supported the assignment of the new species to the genus Chrysogorgia.However,the mtMutS marker showed very limited usefulness for species delimitation and inner relationship inference of Chrysogorgia.In contrast,the 28 S rDNA showed much higher level of genetic variation,and it may be a potential barcode for this genus.In the 28 S rDNA trees,the two new species clustered together.Additionally,compilation of our data showed that 42 of 78(ca.54%) Chrysogorgia species were found in the Indo-West Pacific convergent region,indicating that this area may be a hotspot of deep-water Chrysogorgia species.     

Purification and characterization of an alkaline protease from <Emphasis Type="Italic">Micrococcus</Emphasis> sp. isolated from the South China Sea

Protease is wildly used in various fields, such as food, medicine, washing, leather, cosmetics and other industrial fields. In this study, an alkaline protease secreted by Micrococcus NH54PC02 isolated from the South China Sea was purified and characterized. The growth curve and enzyme activity curve indicated that the cell reached a maximum concentration at the 30th hour and the enzyme activity reached the maximum value at the 36th hour. The protease was purified with 3 steps involving ammonium sulfate precipitation, ion-exchange chromatography and hydrophobic chromatography with 8.22-fold increase in specific activity and 23.68% increase in the recovery. The molecular mass of the protease was estimated to be 25 kDa by SDS-PAGE analysis. The optimum temperature and pH for the protease activity were 50°C and pH 10.0, respectively. The protease showed a strong stability in a wide range of pH values ranging from 6.0–11.0, and maintained 90% enzyme activity in strong alkaline environment with pH 11.0. Inhibitor trials indicated that the protease might be serine protease. But it also possessed the characteristic of metalloprotease as it could be strongly inhibited by EDTA and strongly stimulated by Mn²⁺. Evaluation of matrix-assisted laser desorption ionization/time-of-flight MS (MALDI-TOF-TOF/MS) showed that the protease might belong to the peptidase S8 family.     

Purification and Characterization of an Alkaline Protease from Micrococcus sp.Isolated from the South China Sea

Protease is wildly used in various fields,such as food,medicine,washing,leather,cosmetics and other industrial fields.In this study,an alkaline protease secreted by Micrococcus NH54PC02 isolated from the South China Sea was purified and characterized.The growth curve and enzyme activity curve indicated that the cell reached a maximum concentration at the 30 th hour and the enzyme activity reached the maximum value at the 36 th hour.The protease was purified with 3 steps involving ammonium sulfate precipitation,ion-exchange chromatography and hydrophobic chromatography with 8.22-fold increase in specific activity and 23.68% increase in the recovery.The molecular mass of the protease was estimated to be 25 k Da by SDS-PAGE analysis.The optimum temperature and p H for the protease activity were 50℃ and pH 10.0,respectively.The protease showed a strong stability in a wide range of pH values ranging from 6.0–11.0,and maintained 90% enzyme activity in strong alkaline environment with p H 11.0.Inhibitor trials indicated that the protease might be serine protease.But it also possessed the characteristic of metalloprotease as it could be strongly inhibited by EDTA and strongly stimulated by Mn~(2+).Evaluation of matrix-assisted laser desorption ionization/time-of-flight MS(MALDI-TOF-TOF/MS) showed that the protease might belong to the peptidase S8 family.     

Two new marine benthic diatom species of Hippodonta (Bacillariophyceae) from the coast of China: H. nanjiensis sp. nov. and H. qingdaoensis sp. nov.

The diversity and distribution of Hippodonta species on the coast of China are largely unknown due to limited investigations. In this paper, we report the detailed morphology and ultrastructure of two new species: H. nanjiensis sp. nov., collected from intertidal sandy sediments of Nanji Islands, the East China Sea and H. qingdaoensis sp. nov., collected from intertidal sandy sediments of the No. 1 Bathing Beach in Qingdao City, the Yellow Sea. Both species occur mainly in the middle part of the intertidal zone. H. nanjiensis possesses a unique large round central area. H. qingdaoensis differs from congeners in having a unique stria pattern, wherein the areolae in the middle transapical striae are less developed or absent on the primary side of the valve, and two areolae per stria are apparent under light microscopy(LM). This study expands our knowledge of Hippodonta diversity and distribution on the coast of China.     

Isolation,identification, and cytotoxicity of a new isobenzofuran derivative from marine Streptomyces sp. W007

A new isobenzofuran derivative( 1) was isolated from the marine Streptomyces sp. W007 and its structure was determined through extensive spectroscopic analyses, including 1D-NMR, 2D-NMR, and ESI-MS. The absolute configuration of compound 1 was determined by a combination of experimental analyses and comparison with reported data, including biogenetic reasoning, J-coupling analysis, NOESY, and 1 H- 1 HCOSY. Compound 1 exhibited no cytotoxicity against human cells of gastric cancer BGC-823, lung cancer A549, and breast cancer MCF7.     

Removal of eutrophication factors and heavy metal from a closed cultivation system using the macroalgae, Gracilaria sp. (Rhodophyta)

In this study,the ability of macroalgae Gracilaria sp.of removing eutrophication factors and toxic heavy metals Al,Cr,and Zn in a closed cultivation system is reported.The results show that the concentration of the three heavy metals decreased significantly during the experimental period in an algal biomass dependent manner.The biofiltration capacity of the alga for Al,Cr,and Zn is 10.1%-72.6%,52.5%-83.4% and 36.5%-91.7%,respectively.Using more materials resulted in stronger heavy metal removal.Additionally...