STUDY ON TISSUE CULTURE FOR GELIDIUM SEEDLING

As seedling culture is a crcial factor for successful cultivation of Gelidium, the authors researched tis-sue culture technology for producing seedlings. The merphogeny and experirmental ecology were observedand studied fully in 2-5 mm isoIated tissue fragments, appearaare of branching creepersand attaching structure and new erect seedlings production and development were studied. Fragments weresown on bamboo slice and vinyon rope. The seedlings were cultured 20-30 days indoor, then cultured inthe sea, where the density of erect seedlings was 3- 19 seedlings/cm~2, growth rate was 3.84%/day. The frondarising from seedlings directly was up to 10 cm per year. The ecological conditions for regenerated seedlingsare similar to the natural ones. The regenerated seedlings are sintable for raft culture in various sea areas.     

Formation and early development of tetraspores of Polysiphonia urceolata （Rhodomelaceae, Rhodophyta）

Polysiphonia urceolata is one type of potential commercial red seaweeds used for breeding and cultivation, because of its significant biochemical and biomedical application. However, the information of breeding and seedling incubation for cultivation is limited, especially the early development. In this study, tetrasporohyte and gametophyte of P. urceolata were taken as the study materials in Huiquan Bay, Qingdao, China. The cleaned and sterilized tetrasporophytes and gametophytes were pre-cultured in sterilized seawater, then nurtured at 18°C, 25 μmol photons m⁻² s⁻¹ in 12:12 h (light:dark) photoperiod. Continuous observation under microscope showed that the early development consists of bipolar division stage and seedling stage. In the division stage, tetraspores germinate into bipolar sporelings that further differentiate into a colorless rhizoidal portion and a lightly pigmented upright shoot. The lightly pigmented rhizoidal cell develops to a rhizoid and the larger pigmented cell transforms to an erect axis. In the seedling stage, several quasi-protuberances appear on the erect axis and form juvenile seedlings. The results demonstrate the culture of P. urceolata from tetraspores under laboratory conditions. Supported by National Key Technology Support Program, Development Program of China (No.2006AA09Z21), National Natural Science Foundation of China (No. 40618001 and N_CUHK438/06) and Shandong Agricultural Seed Stock Breeding Project     

The Influences of Mg^2 ,Ca^2 ,and Mg^2 /Ca^2 Ratio in Mixed Seawater on the Emergence Rate of Penaeus Japonicus Postlarva

This paper reports the approprite ranges of Mg^2 ,Ca^2 and their ratio Mg^2 /Ca^2 in mixed seawater for rearing of Penaeus japonicus larvae,The ranges for the above three indices are 1150-1450mg/L,360-440mg/L and 2.8-3.4,respectively,the proper salinity range of mixed seawater is 22.1-33.9 obtained by mixing estuarine water and concentrated seawater.     

Effects of Mg2+, NaCl, citric acid, and other factors on synthesis and accumulation of β-carotene inDunaliella salina

The effect of Mg²⁺, NaCl and citric acid on the accumulation of β-carotene inDunaliella Salina was studied. The experimental results showed that 10.5 mmol/L Mg²⁺, 5 mol/L NaCl, 3 μmol/L citric acid, and CO₂ are favorable forDunaliella Salina cell growth and β-carotene accumulation. After 144 h culture under the above conditions, theDunaliella Salina biomass increased by 7.18 times; β-carotene reached 9.61%. Project 89023990 supported by the Shandong Natural Science Fund.     

The influence of Fe^2＋ on growth and development of cells enzymatically isolated from Porphyra yezoensis blades

Fe^2＋ acted as an accessorial factor for many cellular enzymatic reactions is very important for seaweed growth and development, but the Fe^2＋ requirement in nori had not been seen. Porphyra yezoensis cells were separated enzymatically and cultured in a series of sterilized seawater media containing various concentrations of Fe^2＋. The growth development and cell were investigated in this work. Through this experiment, two biologically-meant concentration scales were found, one is low concentrations, 12.1-102.1μg/L, 10-100 times than that in seawater, favoring the development of isolated cells of Porphyra and the other was high concentrations, more than 10mg/L inhibiting the cell growth, leading to the deformity and shrinkage of the cells. At the concentration of 50 mg/L, the cells stopped growing and died eventually.     

Isolation and cultivation of the vegetative cells ofPorphyra haitanensis

This research deals mainly with the use of the vegetative cells of a marine red algaPorphyra haitanensis (laver) as seeds and their culture into young thalli for further cultivation in the sea. The experimental process was as follows. Enzymatically isolated vegetative cells were attached to rope substrates and cultured in the laboratory for a month until they became about 0.2–0.5 cm long sporelings and were then attached (7–15 sporelings per cm of rope) to nets for removal to the open sea for cultivation. A month after culturing, the thalli reached a maximum length of 50 cm (average of 20–30 cm). The thalli grew faster as the water temperature dropped from 21°C to 17°C. It was proved that the vegetative cells isolated from a small thallus about 5 cm long could grow up into normal thalli after being kept frozen for a year. The results of this study show that vegetative cells can be used as new seeds to simplify the production of laver sporelings. This technique is a significant advance in the field ofPorphyra culture.     

Induction and cultivation of cloned filaments of Polysiphonia urceolata (Rhodomelaceae, Rhodophyta)

A filamentous clone of Polysiphonia urceolata was regenerated from segments cut from the fronds of gametophytes. Unlike wild thalli with short virgate branchlets, the clone was filamentous with few branches. Many transparent trichoblasts arose from pericentral cells during the induction culture, but these were seldom observed during normal growth. The trichoblasts were uniseriate, often colorless, and formed lobed rhizoids rapidly when they came into contact with solid substrates. In addition to morphological characteristics, the photosynthetic properties and growth conditions of the clone differed from those of the mother plant. Cross-gradient light and temperature culture experiments revealed that the most favorable conditions for culture of the filamentous clone were 22°C and 95-120 μE/(m2-s) light intensity. The photosynthetic light saturation value for filaments was approx. 100 μE/(m2-s), which is far lower than that of wild thalli. These results could be used to develop techniques for mass cultures of P. urceolata in photobioreactors for production of seed stock or bioactive products.     

The study on fishing ground of neon flying squid,Ommastrephes bartrami,and ocean environment based on remote sensing data in the Northwest Pacific Ocean

The relationships between the neon flying squid, Ommastrephes bartrami, and the relative ocean environmental factors are analyzed. The environmental factors collected are sea surface temperature (SST), chlorophyll concentration (Chl-a) and sea surface height (SSH) from NASA, as well as the yields of neon flying squid in the North Pacific Ocean. The results show that the favorable temperature for neon flying squid living is 10°C–22°C and the favorite temperature is between 15°C–17°C. The Chl-a concentration is 0.1–0.6 mg/m³. When Chl-a concentration changes to 0.12–0.14 mg/m3, the probability of forming fishing ground becomes very high. In most fishing grounds, the SSH is higher than the mean SSH. The generalized additive model (GAM) was applied to analyze the correlations between neon flying squid and ocean environmental factors. Every year, squids migrate northward from June to August and return southward during October–November, and the characteristics of the both migrations are very different. When squids migrate to the north, most relationships between the yields and SST are positive. The relationships are negative when squids move to southward. The relationships between the yields and Chl-a concentrations are negative from June to October, and insignificant in November. There is no obvious correlation between the catches of squid and longitude, but good with latitude. Supported by the National High Technology Research and Development Program of China (863 Program, No. 2003AA607030); National Key Technology Research and Development Program (No. 2006BAD09A05)     

RELATION OF BODY WEIGHT AND FOOD CONSUMPTION TO METABOLIC RATE OF JUVENILE JAPANESE SEA BASS, LATEOLABRAX JAPONICUS

ＩＮＴＲＯＤＵＣＴＩＯＮＴｈｅＪａｐａｎｅｓｅｓｅａｂａｓｓ (ＬａｔｅｏｌａｂｒａｘｊａｐｏｎｉｃｕｓＣ＆Ｖ)ｄｉｓｔｒｉｂｕｔｅｓｗｉｄｅｌｙｉｎｔｈｅｃｏａｓｔａｌｗａｔｅｒｓａｌｏｎｇｔｈｅＢｏｈａｉＳｅａ ,ＹｅｌｌｏｗＳｅａ ,ＥａｓｔＣｈｉｎａＳｅａ,ａｎｄＳｏｕｔｈＣｈｉｎａＳｅａ .Ａｓａｃａｒｎｉｖｏｒｏｕｓｆｉｓｈ ,ｉｔｏｃｃｕｐｉｅｓｔｈｅｈｉｇｈｔｒｏｐｈｉｃｌｅｖｅｌｉｎｔｈｅｍａｒｉｎｅｅｃｏｓｙｓｔｅｍａｎｄｈａｓａｄａｐｔｅｄｔｏｖａｒｉｏｕｓｅｎｖｉｒｏｎｍｅｎｔａｌｒ…     

FATTY ACIDS PROFILE IN A HIGH CELL DENSITY CULTURE OF ARACHIDONIC ACID-RICH PARIETOCHLORIS INCISA （TREBOUXIOPHYCEAE, CHLOROPHYTA） EXPOSED TO HIGH PFD

The changes in arachidonic acid (AA) and fatty acids profiles along the growth curve ofParietochloris incisa, a coccoid snow green alga, were studied in a 2.8 cm light-path flat photobiorcactor, exposed to strong photon flux density [PFD, 2400 μEmol/(m²·s)]. Sixteen fatty acids were identified by gas chromatography showing that AA was the dominant fatty acid (33%–41%) followed by linoleic acid (17%–21%). AA content was closely investigated with respect to total fatty acids (TFA), ash free dry weight (AFDW) of cell mass as well as total culture content. These parameters were influenced significantly in a similar manner by culture growth phase, i.e., slightly decreasing in the lag period, gradually increasing in the logarithmic phase, becoming maximal at the early stationary phase, starting to decrease at the late stationary phase, sharply dropping at the decline phase. The increase in AA per culture volume during the logarithmic phase was not only associated with the increase in AFDW but also connected with a corresponding increase in AA/TFA, TFA/AFDW as well as AA/AFDW. The sharp decrease in AA content of the culture during the decline phase was mainly due to the decrease in AA/TFA, TFA/AFDW and AA/AFDW, although AFDW declined only a small extent. Maximal AA concentration, obtained at the early stationary phase, was 900 mg/L culture volume, and the average daily net increase of AA during 9 days logarithmic growth was 1.7 g/(m²·day). Therefore, harvesting prior to the decline phase in a batch culture, or at steady state in continuous culture mode seems best for high AA production. The latter possibility was also further confirmed by continuous culture with 5 gradients of harvesting rate. Contribution No. 4138 from the Institute of Oceanology, Chinese Academy of Sciences. Project 39970575 supported by NSFC and A/2786-2 supported by International Foundation for Sciences (IFS).     

ASPECTS OF IRON NUTRITION IN MACROALGAE ULVA PERTUSA （CHLOROPHYTA） UNDER IRON STRESS

Fe, Chlorophyll (Chl) and total nitrogen (TN) content in tissues were measured in Fe-deficient cultures ofUlva. pertusa over a period of 60 days. Photosynthetic carbon fixation rates were studied at the start of and 30 days after Fe-deficiency culture, when the effects of Fe-deficiency on the ultrastructure were also analyzed. The iron content in tissue decreased exponentially during Fe-deficiency (from 726.7 to 31.6 μg/gdw) and simultaneously Chl and TN content declined to 4.35% and 59.9% of their original levels respectively. Maximum carbon fixation rate (50–250 μmol/m² s) under Fe-deficiency decreased significantly compared with the control (p<0.01) and was 13.6 to 0.365 μg C/cm² h. Photosynthesis in Fe-deficient cells became light-saturated at lower irradiance than that in control. Ultrastructural observations of Fe-deficient cells showed reductions in chloroplast number, some degeneration of lamellar organization, an increase in vacuolar area, a decrease in mitochondrial matrix density, and variation in accumulation body number and morphology. During Fe-deficiency, the algae growth rate continued to decline and after 6 weeks of iron deficiency, no further growth was detectable. These suggested that the lower growth rate ofUlva. pertusa under Fe-deficiency could be due mainly to nitrogen utilization and inhibition of photosynthesis. This project No. 39725023 supported by NSFC for Talented Youths and the Project under the Major State Basic Research of China (Grant No. G1999012011).     

FOOD SOURCES AND CARBON BUDGET OF CHINESE PRAWN PENAEUS CHINENSIS

This study deals with contribution of artificial food pellet and natural food to Chinese prawn (Penaeus orientalis) growth in a semiintensive culture pond. The prawn carbon consumption, budget, and the effects of some factors on the budget were investigated. The results showed that 26.2% of P. orientalis growth carbon came from formulated feed at the initial culture stage (when the prawns were 0.06±0.01 g in wet weight), and was 62.5% when the prawns were 9.56±1.04 g. The remaining part of the growth carbon was derived from organic fertilizer and natural food. The highest growth rate occurred at 20×10-3 salinity. Suitable salinity for culturing Chinese prawn was (20-28)×10-3.     

The effects of nitrogen,phosphorus, vitamins and trace metals on the growth of the red tide organismProrocentrum Micans

Laboratory culture experiments showed that <100μ mol/L nitrate, amonium or mixture of amino acids promote the growth of the red tide organismProrocentrum micans Ehrenb, but that >100μmol/L of ammonium, or mixture of glycine and glutamate was harmful to growth, and that orthophosphate wasP. micans’ main phosphorous source in the ocean. Presence of 80μ mol/L EDTA, 0.5 to 1 μmol/L Fe³⁺, 1.0 to 20.0 μ mol/L Mn²⁺ 0.1 to 0.4 μmol/L Co²⁺ in the culture medium could improve the growth ofP. micans. Vitamin B₁ promoted growth, but vitamin B₁₂ and biotin did not. The estimated minimum cell quotas (q _o) for nitrogen and phosphorus being 0.74 pmole/cell and 0.045 pmole/cell show that phosphorus (more than nitrogen) limits the growth ofP. micans in the study area. This project was supported by the Natural Science Foundation of Zhejiang Province.     

DYNAMIC CHANGES OF INORGANIC NITROGEN AND ASTAXANTI—IIN ACCUMULATION IN HAEMATOCOCCUS PLUVIALIS

This study on dynamic changes of culture color,astaxanthin and chlorophylls,inorganic N including N-NO3^-,N-NO2^- and N-NH4^ in batch culture of Haematococcus pluvialis exposed to different additive nitrate concentration showed(1)ast/chl ratio was over 0.8 for brown and red algae,but was usually less than 0.5 for green and yellow algae;(2)N-NO3^-,in general,was unstable and decreased,except for a small unexpected increase in nitrate enriched treatment groups;(3)measurable amounts of N-NO2^- and N-NH4^ were observed respectively with three change modes although no extemal nitrite and ammonia were added into the culture;(4)a non-linear correlation between ast/chl ratio(or color)changes and the levels of N-NO3^-,N-NO2^-,N-NH4^ in H.pluvialis culture;(5)up and down variation of the ast/chl ratio occurred simultaneously with a perceptible color change from yellow to brown(or red)when N-NO3^-,N-NO2^- and N-NH4^ fluctuated around 30,5,5μmol/L respectively;(6)existence of three dynamic modes of N-NO3^-,N-NO2^- and N-NH4^ changes,obviously associated with initial extemal nitrate;(7)the key level of total inorganic N concentration regulating the above physiological changes during indoor cultivation was about 50 μmol/L;and(8)0.5-10mmol/L of nitrate was theoretically conducive to cell growth in batch culture.     

Tolerance of ciliated protozoan Paramecium bursaria （Protozoa,Ciliophora） to ammonia and nitrites

The tolerance to ammonia and nitrites in freshwater ciliate Paramecium bursaria was measured in a conventional open system. The ciliate was exposed to different concentrations of ammonia and nitrites for 2h and 12h in order to determine the lethal concentrations. Linear regression analysis revealed that the 2h-LC50 value for ammonia was 95.94 mg/L and for nitrite 27.35 mg/L using probit scale method (with 95% confidence intervals). There was a linear correlation between the mortality probit scale and logarithmic concentration of ammonia which fit by a regression equation y=7.32x-9.51 (R^2=0.98; y, mortality probit scale; x, logarithmic concentration of ammonia), by which 2 h-LC50 value for ammonia was found to be 95.50 mg/L. A linear correlation between mortality probit scales and logarithmic concentration of nitrite is also followed the regression equation y=2.86x 0.89 (R^2=0.95; y, mortality probit scale; x, logarithmic concentration of nitrite). The regression analysis of toxicity curves showed that the linear correlation between exposed time of ammonia-N LC50 valueand ammonia-N LC50 value followed the regression equation y=2 862.85e-0.08x (R^2=0.95; y, duration of exposure to LC50 value; x, LC50 value), and that between exposed time of nitrite-N LC50 value and nitrite-N LC50 value followed the regression equation y = 127.15e-0.13x (R^2=0.91; y, exposed time of LC50 value; x, LC50 value). The results demonstrate that the tolerance to ammonia in P. bursaria is considerably higher than that of the larvae or juveniles of some metozoa, e.g. cultured prawns and oysters. In addition, ciliates, as bacterial predators, are likely to play a positive role in maintaining and improving water quality in aquatic environments with high-level ammonium, such as sewage treatment systems.     

Bioflocculant Produced by Klebsiella sp. MYC and Its Application in the li＇eatment of Oil-field Produced Water

Seventy-nine strains of bioflocculant-producing bacteria were isolated from 3 activated sludge samples. Among them, strain MYC was found to have the highest and stable flocculating rate for both kaolin clay suspension and oil-field produced water. The bacterial strain was identified as Klebsiella sp. MYC according to its morphological and biochemical characteristics and 16SrDNA sequence. The optimal medium for bioflocculant production by this bacterial strain was composed of cane sugar 20 g L^-1, KH2PO4 2 g L^-1, K2HPO4 5 g L^- 1, （ NH4）2SO4 0.2 g L^-1, urea 0.5 g L^- 1 and yeast extract 0.5 g L^- 1, the initial pH being 5.5. When the suspension of kaolin clay was treated with0.5% of Klebsiella sp. MYC culture broth, the flocculating rate reached more than 90.0% in the presence of 500mg L^-1 CaCI2, while the flocculating rate for oil-field produced water was near 80.0% in a pH range of 7.0 - 9.0 with the separation of oil and suspended particles from the oil-field produced water under similar conditions. The environment-friendly nature of the bioflocculant and high flocculating rate of the strain make the bioflocculant produced by Klebsiella sp. MYC an attractive bioflocculant in oil-field produced water treatment.     

Effects of temperature and irradiance on early development of Chondrus ocellatus Holm (Gigartinaceae, Rhodophyta)

Chondrus is a type of commercially produced red seaweed that widely used for food and carrageen extraction. Although the natural life history of the alga had been well understood, the factors influencing development of the tetraspore and carpospore remain poorly understood. In the perspective of seedling resources, the regulation of early development is crucial for the seedling nursing; therefore, it is necessary to understand the physiological influences during its early development. In this study, we studied the effects of temperature and irradiance on the early development of Chondrus ocellatus Holm under laboratory conditions. The released tetraspores and carpospores were cultivated at different temperatures (10–28°C) and irradiances (10, 60 μmol photons m⁻²s⁻¹) with a photoperiod of 12L:12D. The results indicate that both tetraspores and carpospores are tolerant to temperatures of 10–25°C, and have the highest relative growth rate at 20°C. Irradiance variances influenced the growth of the discoid crusts, and the influence was more significant with increasing temperature; 60 μmol photons m⁻²s⁻¹ was more suitable than 10 μmol photons m⁻²s⁻¹. The optimum temperature and irradiance for the development of seedlings was 20°C and 60 μmol photons m⁻²s⁻¹, respectively.     

Bioaccumulation of Pb<Superscript>2+</Superscript> and its effects on growth,morphology and pigment contents of <Emphasis Type="Italic">Spirulina</Emphasis> (<Emphasis Type="Italic">Arthrospira</Emphasis>) <Emphasis Type="Italic">platensis</Emphasis>

A laboratory experiment was conducted to assess the bioaccumulation of Pb²⁺ and its effects on growth, morphology and pigment contents of Spirulina (Arthrospira) platensis. The specimen cultured in Zarrouk liquid medium was treated with various initial metal concentrations (0, 5, 10, 30, 50 and 100 μg mL⁻¹). The growth of S. platensis was adversely affected by Pb²⁺ at high concentrations (30, 50 and 100 μg mL⁻¹). However, at low concentrations (5 μg mL⁻¹), Pb²⁺ could stimulate its growth slightly. The pigment contents (chlorophyll α and β carotene) were decreased in a dose-dependent manner. The highest reductions (67% and 53% respectively in chlorophyll α and β carotene) were observed in 100 μg mL⁻¹ treatment group. The LC₅₀ (96 h) of Pb²⁺ was measured as 75.34 μg mL⁻¹. Apart from a few cases of filament breakages at elevated concentrations (50 and 100 μg mL⁻¹), morphological abnormalities are not specific. Metal bioaccumulation increased with Pb²⁺ concentrations, but decreased with exposure time. The maximum accumulated amount was 188 mg g⁻¹ dry weight. The bioconcentration factor (BCF) reached to a peak at day 2, followed by a gradual reduction for all the exposure concentrations. S. platensis is able to tolerate considerably high Pb²⁺ concentrations. Consequently it can be used as a potential species to remove heavy metal from contaminated waters.     

Enhanced carotenoid production by a mutant of the marine yeast Rhodotorula sp. hidai

After a serial of UV, EMS and NTG mutagenesis, a mutant named MM of the red marine yeast strain Rhodotorula sp. hidai was obtained. The mutant MM could produce 603.93 μg g⁻¹ of carotenoid within 5 days in the medium containing 4.0 g sucrose, 1.5 g yeast extract, 0.1 g MgSO₄, and 100 mL of sea water, with pH 6.0 and at 30 °C, while only 213.18 μg g⁻¹ of carotenoid was produced by the wild type under the same conditions.