4株形态不同的长心卡帕藻(Kappaphycus alvarezii)的初步ISSR分析

为了研究高产胶量品系的遗传学特征,建立了长心卡帕藻(Kappaphycus alvarezii)物种的ISSR分析方法。用ISSR技术对4株同一来源,形态各异的长心卡帕藻进行了遗传学分析,与11条ISSR引物共扩增得到60条带,其中1条带具有多态性并推测与稳定的经济性状连锁。说明ISSR技术可应用于长心卡帕藻品系筛选,结合其他手段,为分子标记辅助育种提供了可能。

Molecular identification based on ITS sequences for Kappaphycus and Eucheuma cultivated in China

The systematic classification of the Eucheumatoideae is difficult because of their variable morphology and interpretation of reproductive structures. Kappaphycus and Eucheuma specimens cultivated on the Hainan and Fujian coast of China were introduced from Vietnam, the Philippines and Indonesia. Combined with morphological characteristics, all Kappaphycus and Eucheuma cultivated strains were identified by internal transcribed spacer (ITS) sequences. The phylogenetic tree was constructed using neighbor-joining and maximum likelihood methods. The results indicate that different ITS sequence lengths occurred in the different genera and species. An obvious difference in morphology could be found in the protuberance shape between Kappaphycus and Eucheuma. The protuberance in Eucheuma was thorn-like and in Kappaphycus was wartlike or papillate. Their ITS sequence lengths differed significantly in nucleotide variation rates up to 58.55%-63.90%. All nucleotide variations occurred in the ITS1 and ITS2 regions except for five nucleotide transversions in the 5.8S rDNA region. In addition, the difference was at the branches among congeneric species. Kappaphycus sp. had branches with small buds, while K. alvarezii did not have such a feature. The nucleotide variation rates varied from 7.02% to 7.48% among species; within the same species of the clades it was <1.20%. Eucheumatoideae algae cultivated in China consisted of three clades, K. alvarezii, Kappaphycus sp., and E. denticulatum. The results indicate that ITS sequence analysis was an effective way for identification of interspecies and intraspecies phylogenetic relationships and might provide a clue for molecular identification of algal Eucheumatoideae.

Studies on the isolation and culture of protoplasts from Kappaphycus alvarezii

In this study, protoplasts were successfully isolated from Kappaphycus alvarezii using snail enzymes, abalone enzymes and cellulase. The optimum enzymic ratio was fixed to be 20% of abalone enzyme, 12% of cellulase and the osmotic stabilizer was 2.0 mol/L glucose. The optimum enzymic hydrolysis conditions were found to be dark enzymolysis at 30°C continuing for 4.0 h. The resultant density and yield of protoplasts achieved 32.60×10^4 mL-1, 65.20×10^4 g-1 tissue for Kappaphycus alvarezii. Finally, under the temperature of 20°C, light intensity of 1 500–2 000 lx and photoperiod of 12 h/d, two developmental pathways were investigated：（1） callus-like cell mass and regenerated plantlet occurred on protoplast;（2） young shoots and calluslike cell mass occurred in tissue blocks after enzymolysis.

Assessment of introduced Kappaphycus (Solieriaceae, Rhodophyta) species relationships in China with molecular markers

Due to morphological plasticity and paucity of diagnostic morphological characters, the taxonomy of Kap- paphycus gets more and more confused with the expanding of commercial cultivation. In this study, the phylogenetic relationship of 13 strains of introduced Kappaphycus species in China was defined using DNA molecular markers, such as 18S rDNA, rbcL and cox2-cox3 spacer region. The resolutions obtained by three different molecular markers were compared： both cox2--cox3 spacer region and rbcL sequences are eligible in inter- species identification of Kappaphycus, whereas cox2-cox3 spacer region is more variable than rbcL sequence. There is several basepairs＇ discrepancy among 18S rDNA sequences, while it is 100% identical among both cox2-cox3 spacer region and rbcL sequences of the ten strains of K. alvarezii. We suppose that 18S rDNA sequence can provide more information in biogeography study of Kappaphycus than other two DNA sequences.

Sauna Technique, Drying Kinetic Modeling and Effectiveness on Solar Drying Compared with Direct Drying in Drying Process of <i>Kappaphycus striatum</i>in Selakan Island Malaysia

A sauna drying technique—the solar drier was designed and imposed, constructed and tested for drying of seaweed. The seaweed moisture content was decreased around 50% in 2-day sauna. Kinetic curves of drying of seaweed were known to be used in this system. The non-linear regression procedure was used to fit three different drying models. The models were compared with experimental data of red seaweed being dried on the daily average of air temperature about 40℃. The fit quality of the models was evaluated using the coefficient of determination (R2), Mean Bias Error (MBE) and Root Mean Square Error (RMSE). The highest values of R2 (0.99027), the lowest MBE (0.00044) and RMSE (0.03039) indicated that the Page model was the best mathematical model to describe the drying behavior of sauna dried seaweed. The percentage of the saved time using this technique was calculated at 57.9% on the average solar radiation of about 500 W/m2 and air flow rate of 0.056 kg/s.

Comparison study of anti-microbial activity between crude extract of Kappaphycus alvarezii and Andrographis paniculata

To evaluate the antimicrobial properties of Kappaphycus alvarezii (K. alvarezii) and Andrographis paniculata (A. paniculata) and to compare the microbial inhibition activities between these two crude extracts.MethodsBoth K. alvarezii and A. paniculata were extracted with methanol before the commencement of antimicrobial properties studies. There were a total of eight species of bacteria, including Gram-positive bacteria Bacillus cereus, Bacillus subtilis, Staphylococcus aureus and Staphylococcus epidermidis and Gram-negative bacteria, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Salmonella enterica. The antimicrobial activity was tested by disk diffusion method.ResultsCrude extract of K. alvarezii was found not effective against both Gram-positive and Gram-negative bacteria. In contrast, A. paniculata showed higher inhibition towards the growth of Gram-positive bacteria compared to Gram-negative bacteria. Results revealed that Bacillus subtilis was susceptible at lower concentration of A. paniculata crude extract however Staphylococcus epidermidis was the most susceptible towards A. paniculata at higher concentration. Although the inhibition zones produced by the crude extract were smaller than that of the positive control, streptomycin disc, A. paniculata crude extract still can be considered as potential antimicrobial agents either because it is a natural product or the active compound which is yet identified from its crude extract.ConclusionsCrude extract of K. alvarezii has zero inhibition in bacteria growth whereas A. paniculata exerted higher inhibition towards Gram-positive bacteria. The bioactive compounds contained by A. paniculata can be evaluated in order to yield a better vision towards the mode of action.

Growth Rate of Kappaphycus striatum var cottonii at Different Positions of the Cultivation Lines to the Direction of Water Current

The study was conducted to determine the effects of different positions in cultivation lines towards the direction of water current on growth rate of Kappaphycus striatum var. cottonii for a duration of 45 d at 4°30′9.36″ N and 119°22′6.8″ E of Bungin Pondohan, Sitangkai, Tawi-Tawi. The experiment has three treatments;the parallel (T1), perpendicular (T2) and diagonal (T3) positions towards the direction of water current with five replicates in each treatment following the randomized complete block design (RCBD). Results showed that higher mean weight of 473 g and growth increment of 8.3 g/d were obtained from T3, although not significantly different from T1 and T2 with 431 g and 7.4 g/d, and 408 g and 6.8 g/d, respectively (p>0.05). However, results showed statistically similar growth rates of K. striatum var cottonii among all treatments. Thus, the positions of cultivation lines with respect to the direction of the current have no significant effect on the growth of the said seaweed species. Hence, the treatments used are viable in growing K. striatum var. cottonii in the selected study site.

Development of Novel Bread by Combining Seaweed Kappaphycus alvarezii from Sri Lanka and Saccharomyces cerevisiae Isolated from Nectarine

Seaweeds are not used as much for edible use although many kinds of seaweed are collected in Sri Lanka.The objective of this study was to develop high quality bread using seaweed,Kappaphycus alvarezii collected from Sri Lanka,and wild yeast,Saccharomyces cerevisiae isolated from nectarine in Japan.Dietary fiber in K.alvarezii,Japanese sea weeds,Wakame and Aosa contained 48.1,31.7 and 29.1 g/100 g,respectively.The amount of total organic acid increased in garlic bread compared to bread without garlic.The best swelling properties were evident in the bread with K.alvarezii(0.5%)and garlic.The bread including K.alvarezii increased the fermentative power of wild yeast while the addition of garlic increased the degree of swelling of the dough.In the sensory testing,the bread with K.alvarezii(0.5%)and garlic was favored for the fineness of its texture and had the most preferred texture.It is possible to develop seaweed bread that is rich in dietary fiber and has excellent flavor by adding a suitable amount of garlic during the fermentation process.

Enhanced Yield and Quality in Some Banana Varieties Applied with Commercially Manufactured Biostimulant Aquasap from Sea Plant Kappaphycus alvarezii

A field study was conducted to investigate the effect of Aquasap, a commercially manufactured biostimulant from seaweed Kappapphycus alvarezii on some hills and foothills banana varieties namely Robusta （AAA）, Njali poovan （AB）, Red banana （AAA） and Nendran （AAB） by applying at 5% through foliar spray at the 3rd, 5th and 7th month of plantation. Both quantitative and quality data of fruits were analyzed along with vegetative growth of the plants. The seaweed biostimulant was found very effective on increasing the weight and nutritional contents of all four banana varieties tested. The highest yield was found in Robusta with 56.58% over control followed by Njali poovan, Red banana and Nendran with 19.08%, 39.35% and 11.46%, respectively. Yield of fruits per hectare of four varieties treated were 76.96, 19.23, 29.68 and 23.37 metric ton （mt）, respectively. Fruits of treated plants also showed less moisture level as compared to control with 240.61%, 62.30%, 61.68% and 37.18% more carbohydrate, 283.71%, 94.07%, 62.87% and 38.93% higher proteins and 153.02%, 47.37%, 58.73% and 31.48% higher minerals respectively as compare to control plants. Therefore, the simple practice of application of eco-friendly seaweed based biostimulant would benefit small and marginal farmers of banana.

Antiproliferative Activity of Kappaphycus Alvarezii Extract on Three Cancer Cell Lines （NCIH 460, HCT 116 and U 251）

The methanolic extract of red seaweed Kappaphycus alvarezii was evaluated against three different cancer cell lines to study for its antiproliferative effect. Lung cancer cell line （NCIH 460）, Colon cancer cell line （HCT 116） and Glial cell carcinoma （U 251） are the three selected cell lines investigated in this study. Different concentrations of methanol extract （0.01, 0.10, 1.00, 10.00 and 100.00 lag/mL respectively） of Kappaphycus alvarezii were prepared and screened by quantitative MTT （Microculture Tetrazolium） （3-（4, 5-dimethylthiazol）-2, 5-diphenyltetrazolium bromide） assay. MTT assay are the colorimetric assay which was applied to assess the viability and proliferation of cancer cells to determine the cytotoxicity of methanol extract ofK. alvarezii. The MTT test is based on the enzymatic reduction of the tetrazolium salt （MTT） to formazon crystals exclusively in living metabolically active cells developed purple color complex which was directly proportional to the viability of cells. To elucidate the in-vitro anticancer activity the Lethal Concentration （LC50）, Growth Inhibition （GI50） and Total Growth Inhibition （TGI） of the extract were investigated individually for each cancer celt line. Analysis of the extract has shown good cytotoxicity in all tested cancer cell lines, with an IC50 of 55 μg/mL against NCIH460 and U251, 65 μg/mL for HCT116 respectively. GI50 was found to be 5 μg/mL for NCIH 460 and 10 μg/mL for HCT 116 and U251 cell lines. TGI was 19 μg/mL for NCIH 460, 29 μg/mL for HCT 116 and 25 μg/mL for U 251 cell lines. The cytotoxicity of the extract were significantly high in Lung Cancer Cell line （NCIH 460） when compared to Colon Cancer Cell line （HCT 116） and Glial Cell Carcinoma （U251） in the following order NCIH 460 〉 HCT 116 〉 U251.

Nutrient and Nutraceutical Potentials of Seaweed Biomass Ulva lactuca and Kappaphycus alvarezii

The study aimed at evaluating the nutrient and nutraceutical potentials of the selected seaweed biomass namely Ulva lactuca and Kappaphycus alvarezii. The selected seaweeds were found to be rich in macro and micronutrients. Kappaphycus contained high amount of ash when compared to U. lactuca. It is revealed that the mercury level in U. lactuca was 0.017 ppm and it was not detected in K. alvarezii. The heavy metal concentration in the selected seaweeds was within the tolerable value reported as quality criteria for edible seaweeds. These seaweeds were also found to be rich sources of vitamin antioxidants namely Vitamin C, total carotenoid, beta carotene, total chlorophyll and polyphenol. The phytochemical screening of the selected seaweeds showed that both seaweeds contained carbohydrates, protein, gums and mulicage, phenols, starch and quinones. Alkaloids was absent in Kappaphycus, saponins was moderate in U. actuca when compared to Kappaphycus. Glycosides, flavonoids, tannins and terpenoids were absent in both the species. The selected seaweeds also possess antibacterial activity. The selected biomass could be recommended for use in dietaries to combat protein energy malnutrition and in particular micronutrient deficiencies.

Kappaphycus alvarezii waste biomass:A potential biosorbent for chromium ions removal

The Cr（III） sorption experiments onto Kappaphycus alvarezii waste biomass were conducted at different pH values（2-6） under the conditions of initial metal concentration of 10-50 mg/L and the chemical compositions of Cr-Cu and Cr-Cd.The Cr（III） sorption capacities were slightly dependent on pH,and the maximum sorption capacity was 0.86 mg/g at pH 3.The sorption capacities increased with increase in the initial metal concentration,whereas it was suppressed by the presence of Cu（II） and Cd（III） in the solution.The Cr（III） sorption equilibrium was evaluated using Langmuir,Freundlich and BET isotherms.The sorption mechanisms were characterised using scanning electron microscopy and Fourier transform infrared spectroscopy.The main mechanisms were ion exchange coupled with a complexation mechanism.Kappaphycus alvarezii waste biomass represents a potential for Cr（III） ion removal from aqueous solution.

光照强度和盐度对长心卡帕藻生长的影响

在室内采用静置培养法研究了光照强度和盐度对红色和绿色品系长心卡帕藻Kappaphycus alvarezii生长的影响,主要测定了不同光照强度(30、65、110、150和200μmol.(m2.s)-1)和不同盐度(23、28、32、35和41)对这2种藻的生长率、成活率、色素含量及其吸收光谱的影响。红色品系长心卡帕藻适宜的光照强度范围为30—65μmol.(m2.s)-1,光照强度为30μmol.(m2.s)-1时培养,其成活率和日生长率都最高,分别达100%和1.25%,强光下培养生长率和成活率明显下降;红色品系长心卡帕藻适宜的盐度范围为28—32,最适为32。绿色品系长心卡帕藻适宜150—200μmol.(m2.s)-1的强光,150μmol.(m2.s)-1光照强度下培养日生长率最高,达1.05%,各种光照强度下培养其成活率变化不大;绿色品系长心卡帕藻适宜的盐度范围为23—32,最适为23。不同光照强度和盐度条件下培养,长心卡帕藻体内的色素含量会发生变化,而色素含量与其生长率有或没有明显的相关关系。不同光照强度、盐度条件下培养,藻体内的色素吸收光谱特性不变。以上研究结果对我国长心卡帕藻的栽培生产具有一定的指导意义。

篮子鱼内脏粗提液制备长心卡帕藻和细齿麒麟菜原生质体的初步研究

于2011年9月至2013年1月,在海南陵水热带海藻实验基地,利用篮子鱼（Siganus fuscessens）内脏研磨制备消化酶,开展了酶解制备大型产胶红藻长心卡帕藻（Kappaphycus alvarezii）和细齿麒麟菜（Eucheuma denticulatum）的原生质体研究,观察了不同酶解条件对两种产胶海藻原生质体产量和产率的影响。结果表明,细齿麒麟菜原生质体得率高于长心卡帕藻;在藻泥与酶浓度配比中,长心卡帕藻或细齿麒麟菜藻泥越多,所制备出的原生质体产量越高,而其制备效率却越低;在温度（20-30℃）梯度中,有最高原生质体产量和效率的酶解温度为25℃;在pH 6.0-8.0的实验酶解梯度中,酶解pH 6.0时的酶解效果最好;在12-48 h实验酶解时间梯度中,酶解时间为48 h时原生质体产量和得率最高;酶解时间越长,长心卡帕藻或细齿麒麟菜原生质体制备效率越高。为此建议在25℃和pH6.0条件下,适当增加海藻藻泥用量和延长酶解时间,以更有效获得原生质体。

De novo sequencing and comparative analysis of three red algal species of Family Solieriaceae to discover putative genes associated with carrageenan biosysthesis

Betaphycus gelatinus, Kappaphycus alvarezii and Eucheuma denticulatum of Family Solieriaceae, Order Gi-gartinales, Class Rhodophyceae are three important carrageenan-producing red algal species, which pro-duce different types of carrageenans, beta （β）-carrageenan, kappa （κ）-carrageenan and iota （ι）-carrageenan. So far the carrageenan biosynthesis pathway is not fully understood and few information is about the So-lieriaceae genome and transcriptome sequence. Here, we performed the de novo transcriptome sequencing, assembly, functional annotation and comparative analysis of these three commercial-valuable species using an Illumina short-sequencing platform Hiseq 2000 and bioinformatic software. Furthermore, we compared the different expression of some unigenes involved in some pathways relevant to carrageenan biosynthe-sis. We finally found 861 different expressed KEGG orthologs which contained a glycolysis/gluconeogenesis pathway （21 orthologs）, carbon fixation in photosynthetic organisms （16 orthologs）, galactose metabolism （5 orthologs）, and fructose and mannose metabolism （9 orthologs） which are parts of the carbohydrate me-tabolism. We also found 8 different expressed KEGG orthologs for sulfur metabolism which might be impor-tantly related to biosynthesis of different types of carrageenans. The results presented in this study provided valuable resources for functional genomics annotation and investigation of mechanisms underlying the biosynthesis of carrageenan in Family Solieriaceae.

Assessment of Nutrients in Seaweed Tank from Land Based Integrated Multitrophic Aquaculture Module

Aquaculture industry is often generating waste that negatively impact to the environment. These wastes are rich in nutrients. Seaweed can utilize these waste nutrients. This experiment was conducted in a flow-through system (FTS) and a recirculation system (RS) in land-based integrated multi-trophic aquaculture module using seaweed Kappaphycus alvarezii as one of the components. The aim was to access the efficiency of the removal of nutrients in the integrated culture. Kappaphycus alvarezii was stocked at the density of 1 kg/tank in the rectangular plastic tank with 500 L of seawater. The waste generated from integrated multi-trophic aquaculture tanks was directed towards K. alvarezii culture tank. Water samples from inlet, outlet and inside of the culture tanks were drawn to determine the nutrients, namely, total nitrogen (mg/L) and total phosphorus (mg/L). Total nitrogen and total phosphorus in the seaweed sample and from the sediment of culture tanks were also analyzed. The total nitrogen amounting to 59.5% and 61.6% nitrogen was taken up by K. alvarezii in FTS and RS culture tank, respectively. The phosphorus showed the highest deposition of 61.1% and 31.6% in the sediment of in FTS and RS culture tanks respectively, whereas only 5.5% and 3.4% of phosphorus were taken up by K. alvarezii from FTS and RS culture tanks, respectively. The percentage of nitrogen remained in water was comparatively higher by 14.2% and 27.5% than phosphorus by 8.3% and 23.0% in water of both FTS and RS culture tanks, respectively. These results indicated that this species seaweed is efficient in the removal of nitrogen from both FTS and RS culture tank.

长心卡帕藻两种颜色藻株的生长差异研究

分别研究了长心卡帕藻(Kappaphycus alvarezii)的两种颜色藻株在不同月份和不同养殖方式下的生长差异。结果表明,长心卡帕藻红色藻株在3月份的相对生长率为(2.52±0.31)%·d-1,显著高于绿色藻株(P<0.05);绿色藻株在4月份的相对生长率为(6.51±0.58)%·d-1,显著高于红色藻株(P<0.05);5月份两种颜色藻株生长无显著差异。不同养殖方式的生长对比结果显示,两种颜色藻株在浮绳吊养方式下能获得更高的生长速度,红色藻株的相对生长率在浮绳吊养方式下显著高于网片挂养方式(P<0.05),但网片挂养方式具有节省劳力和方便推广等特点。