A BROAD SPECTRUM ARTIFICIAL SEA WATER MEDIUM FOR COASTAL AND OPEN OCEAN PHYTOPLANKTON1

A new artificial seawater medium has been tested with 83 strains of coastal and open ocean phytoplankton from 11 different algal classes. The cultures were carried through four transfers, representing a period of eight weeks for most species. Only three species could not be maintained in the enriched artificial seawater, and 16 species, mainly from the Prymnesiophyceae and Dinophyceae, had reduced final cell yields compared to those grown in enriched natural seawater. Since 77% of the species tested grew equally well in enriched artificial or natural seawater and more than 95% could be maintained in the artificial medium, this recipe is useful over a broad spectrum of species. The artificial seawater base was enriched with a modified ES enrichment solution; the primary modifications were the omission of Tris and the addition of Si. Enriched medium was autoclaved without precipitation by lowering the pH before autoclaving. This was accomplished by adding equimolar amounts of Na-HCO₃ and HCl which produced NaCl and CO₂ during the heating process. When no pH buffer was used, precipitation could only be avoided by autoclaving the artificial seawater base as two separate salt solutions (with Ca and Sr separated from CO₃^?2 and SO₄^?2), cooling, mixing and aseptically adding the sterilized enrichment solution.     

EVOLUTION OF AN ARTIFICIAL SEAWATER MEDIUM: IMPROVEMENTS IN ENRICHED SEAWATER, ARTIFICIAL WATER OVER THE LAST TWO DECADES

Although most phycologists use natural seawater for culturing marine species, artificial media continue to play important roles in overcoming problems of supply and seasonal variability in the quality of natural seawater and also for experiments involving manipulation of micro- and macronutrients. Several artificial media have been developed over the last 90 years; enriched seawater, artificial water (ESAW) is among the more popular recipes. ESAW has the advantage of an ionic balance that is somewhat closer to that of normal seawater. The original paper compared the growth of 83 strains of microalgae in natural seawater (ESNW) versus ESAW and determined that 23% grew more poorly in the artificial water. Since 1980, however, the composition of ESAW, as used by the original authors, has changed considerably. In particular, the added forms of phosphate, iron, and silicate have been changed and the trace metal mixture has been altered to include nickel, molybdenum, and selenium. We tested whether these changes improved the ability of the artificial medium to grow previously difficult to grow phytoplankton species. To test this, we selected eight species that had been shown to grow better in ESNW than in ESAW and compared their growth again, using the currently used recipe with all the above modifications. For all but one species ( Apedinella spinifera ), growth rate and final yield was no different between the media but in one case ( Emiliania huxleyi ) was slightly higher in ESAW. No differences in cell morphology or volume were found in any case. We conclude that changes to the enrichment portion of the recipe have significantly improved this artificial seawater medium and that it can be used to grow an even wider range of coastal and open ocean species.     

Disposable bioreactor for cell culture using wave-induced agitation

This work describes a novel bioreactor system for the cultivation of animal, insect, and plant cells using wave agitation induced by a rocking motion. This agitation system provides good nutrient distribution, off-bottom suspension, and excellent oxygen transfer without damaging fluid shear or gas bubbles. Unlike other cell culture systems, such as spinners, hollow-fiber bioreactors, and roller bottles, scale-up is simple, and has been demonstrated up to 100 L of culture volume. The bioreactor is disposable, and therefore requires no cleaning or sterilization. Additions and sampling are possible without the need for a laminar flow cabinet. The unit can be placed in an incubator requiring minimal instrumentation. These features dramatically lower the purchase cost, and operating expenses of this laboratory/pilot scale cell cultivation system. Results are presented for various model systems: 1) recombinant NS0 cells in suspension; 2) adenovirus production using human 293 cells in suspension; 3) Sf9 insect cell/baculovirus system; and 4) human 293 cells on microcarrier. These examples show the general suitability of the system for cells in suspension, anchorage-dependent culture, and virus production in research and GMP applications. This revised version was published online in July 2006 with corrections to the Cover Date.     

Production characteristics of <Emphasis Type="Italic">Phaeodactylum tricornutum</Emphasis> Bohlin grown on medium with artificial sea water

The growth of marine diatoms Phaeodactylum tricornutum was investigated on a medium with artificial sea water under artificial and natural light. The maximum specific growth rate was 0.7 day^–1, the productivity was 0.8 g/L day, and the maximum biomass was 3.86 g/L under artificial light in laboratory conditions. In the conditions of Crimea, the maximum productivity of P. tricornutum amounted to 6 g/m² day under natural light in an outdoor photobioreactor (pool). The results of cultivation of P. tricornutum in a pool with artificial seawater under natural light may serve as a basis for developing technologies for the industrial cultivation of algae.     

Environmental Factors that influence the Transition from Lysogenic to Lytic Existence in the ϕHSIC/Listonella pelagia Marine Phage–Host System

The marine phage ϕHSIC has been previously reported to enter into a pseudolysogenic-like interaction with its host Listonella pelagia. This phage–host system displays behaviors that are characteristic of both pseudolysogeny and lysogeny including a high rate of spontaneous induction and chromosomal integration of the prophage. To determine what parameters may influence the transition from lysogenic to lytic existence in the ϕHSIC/L. pelagia phage–host system, cultures of this organism were incubated under different environmental conditions, while host cell growth and bacteriophage production were monitored. The environmental parameters tested included salinity, temperature, a rapid temperature shift, and degree of culture aeration. The highest titers of phage were produced by HSIC-1a cells grown in high-salinity nutrient artificial seawater media (67 ppt with a natural salinity equivalent of 57 ppt) or those cultured in highly aerated nutrient artificial seawater media (cultures shaken at 300 rpm). Conversely, the lowest titers of phage were produced under low salinity or rate of aeration. In general, conditions that stimulated growth resulted in greater lytic phage production, whereas slow growth favored lysogeny. These results indicate that elevated salinity and aeration influenced the switch from lysogenic to lytic existence for the phage ϕHSIC. These results may have implications for environmental controls of the lysogenic switch in natural populations of marine bacteria.     

The influence of microamounts of organic substances other than vitamins on the growth of some red algae in axenic culture

Several red algae in axenic culture decline in artificial seawater after a period of cultivation. Nemalion helminthoides rapidly loses its brown-red colour and grows with longer and thinner threads. Extracts of contaminated red algae, or nutrient medium in which they have grown, as well as fresh seawater from the Fucus-Ascophyllum zone, enhance growth and restore the pigmentation of the cells. Addition of 40 mg Bacto-Casamino acids per litre of nutrient medium had the same effect. After fractional precipitation with ethyl alcohol and separation on a Sephadex column the activity was localised to a peptide fraction. Results from chromatography point to effects from a low molecular weight substance active alone or coupled to a peptide.     

Improved algal oil production from Botryococcus braunii by feeding nitrate and phosphate in an airlift bioreactor

To improve biomass and microalgal oil production of Botryococcus braunii, fed‐batch culture was investigated in an airlift photobioreactor. The optimal feeding time of the fed‐batch culture was after 15 days of cultivation, where 1.82 g/L of the microalgal biomass was obtained in the batch culture. Nitrate nutrient was the restrictive factor for the fed‐batch cultivation while phosphate nutrient with high concentration did not affect the microalgal growth. The optimal mole ratio of nitrate to phosphate was 34.7:1, where nitrate concentration reached the initial level and phosphate concentration was one quarter of its initial level. With one feeding, the biomass of B. braunii reached 2.56 g/L after 18 days. Two feedings in 2‐day interval enhanced the biomass production up to 2.87 g/L after 19 days of cultivation. The hydrocarbon content in dry biomass of B. braunii kept at high level of 64.3% w/w. Compared with the batch culture, biomass production and hydrocarbon productivity of B. braunii were greatly improved by the strategic fed‐batch cultivation.     

A LOW-VOLUME FLOW TANK FOR MEASURING NUTRIENT UPTAKE BY LARGE MACROPHYTES1

A recirculating flow-tank was designed and tested to measure inorganic nutrient uptake by, and visualize the movement of seawater around, large macrophytes. The tank volume was small (46 L), and a propeller drive produced unidirectional mean flow. A turbulence reduction section dampened turbulence in the test section to a low level, so that water movement within this region was virtually laminar. The test section of the tank was wider than that of previous designs, allowing whole blades of Macrocystis integrifolia Bory and juvenile plants of Nereocystis luetkeana (Mert.) Post, et Rupr. to be placed in the flow, away from the influence of velocity boundary layers associated with the tank walls. The tank's use in macroalgae nutrient uptake and flow visualization experiments was demonstrated.     

Responses of benthic algal communities and their traits to experimental changes in fine sediments,nutrients and flow

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Growth ofEnteromorpha linza in sewage effluent and sewage effluent-seawater mixtures

Enteromorpha linza (L.) J. Ag. was grown in full strength sewage effluent, various combinations of sewage effluent and seawater, and in natural seawater. It was found that full strength sewage effluent with a salinity of 14 supported best growth of the alga. After a 12 day cultivation period, growth ofE. linza in full strength sewage effluent and 75% sewage effluent- seawater mixture showed 3.5-fold and 2-fold increase in fresh weight over that grown in natural seawater; respectively. Uptake of PO _inf4 ^sup3– -P, NH₃-N and NO _inf3 ^sup– -N by cells ofE. linza was extremely efficient in all tested media. Data obtained from the experiments indicated that inorganic nitrogen rather than phosphorus was the limiting nutrient factor for growth ofE. linza in full strength sewage effluent and in other sewage effluent- seawater mixtures. NH₃-N at concentrations above 4.5 ppm was found to inhibit uptake of NO _inf3 ^sup– -N in the same culture medium by the algal cells. The fact that sewage grownE. linza contained comparatively much higher protein content (30.2% dry weight) than that grown in natural seawater (12.5% dry weight) leads to the conclusion that sewage grownE. linza could serve as an economically feasible feed for livestock in Hong Kong where the sewage is characterized by having a salinity of approximately 14. It is proposed that this multicellular green alga is a suitable algal species to serve the dual function of wastewater purification through the production of algal protein from sewage effluent having high salinities.     

On the cultivation of free-living marine and estuarine nematodes

Although a large body of literature exists on the systematics and ecology of free-living marine and brackish-water nematodes, key questions on the nature and magnitude of interactions between nematodes and other organisms in the benthos remain unanswered. Relatively few authors have investigated live nematodes in food web studies or in experiments dealing with the nematodes’ response to a varying environment. It is mainly for the latter purpose that attempts have been made to maintain, rear and cultivate selected species. This paper describes the methodology used for the maintenance, rearing, and eventual permanent agnotobiotic cultivation of a variety of estuarine nematodes. Spot plates, where small samples of sediment or macrophyte material are inoculated on a sloppy agar layer, have been used for the purpose of maintenance and initial cultivation. Those species that reproduce on spot plates are then selected for monospecific cultivation on agar layers with different nutrient enrichments and with micro-organisms cotransferred from the spot plates as food. Mixtures of bacto and nutrient agar prepared in artificial seawater were specifically suitable for the xenic cultivation of nine bacterivorous and, when supplied with Erdschreiber nutrients, two algivorous/bacterivorous nematode species. Up to three generations of five other nematode species have been reared under laboratory conditions, and several more were kept alive and active for variable periods of time on agar. Generation times observed on spot plates forAdoncholaimus fuscus andOncholaimus oxyuris were substantially shorter than previously published estimates and suggest a correspondingly higher predatory and scavenging potency for these and related enoplids. A procedure for the long-term storage of nematodes at −80°C with glycerol as a cryoprotectant was successfully used forDiplolaimella dievengatensis, Panagrolaimus sp. 1, andPellioditis marina, but not forDiplolaimelloides meyli. The authors have also summarized the existing literature on the cultivation of marine and brackish-water nematodes. Continuous cultivation appears to have been successful mainly for Aufwuchs and epiphytic nematodes; only few sediment-dwellers have been established in permanent culture. Of only just over 30 species that have ever been cultivated, more than half belong to one family (Monhysteridae) and three are Rhabditida, an order poorly represented in the marine environment. Four species have been grown in monoxenic and one in axenic culture, the latter though with limited success. It is concluded that our understanding of the basic nutritional requirements of marine nematodes is as yet insufficient, and that the culture techniques which have so far mainly deployed agar or liquid substrates, while being suitable for the cultivation of Aufwuchs and epiphytic nematodes, do not accurately enough mimic gradients specific of the natural habitat of many sediment-dwellers.     

基于人工培养的浮萍快速无性繁殖营养条件研究

为探究并优化浮萍人工培养技术, 研究以广布种紫萍(Spirodela polyrrhiza)和青萍(Lemna minor)为主要研究对象, 探索两种浮萍植物在不同营养水平的Hoagland和Hunter培养液中的鲜重、叶状体数的生长变化状况。结果表明: (1)紫萍和青萍鲜重在Hoagland培养液的不同营养水平下先增加后减少, 而在Hunter营养液中呈持续增长趋势; 两种浮萍的鲜重最大相对增长率(RGR)分别为0.11和0.18, 鲜重受浮萍品种和培养基类型及其不同营养水平影响显著(P<0.05), 以青萍在Hunter原液培养基下的无性繁殖产生的生物量最高。(2)紫萍和青萍叶状体数在Hoagland培养液的不同营养水平下先增加后减少, 而同鲜重一样在Hunter营养液中呈不断增长趋势; 两种浮萍的叶状体最大相对增长率分别为0.14和0.19, 叶状体生长的RGR变化同样受浮萍品种和培养基类型及营养水平影响显著(P<0.05), 以青萍在Hunter原液的营养环境下收获的叶状体数最高。(3)两种浮萍在Hoagland和Hunter营养液的不同营养水平下鲜重/叶状体比呈下降趋势, 表明两种浮萍在适应不同营养时优先繁殖子代叶状体, 以扩大种的适合度。研究认为Hunter原液可作为广布种青萍的最优培养条件, 可实现短时间内收获较大浮萍鲜生物量和叶状体数, 为进一步资源化利用提供原材料。     

Food and specific macronutrient limitation in an assemblage of predatory beetles

Habitats vary in food resources with carnivores often being prey limited, but it is unclear whether habitats facilitate a nutritionally balanced diet. Two paradigms in nutritional ecology, ecological stoichiometry and nutritional geometry, predict that carnivores are limited mainly by protein or lipid, respectively. Using the carabid beetle Anchomenus dorsalis and 10 other predatory beetles from agricultural fields, we developed and tested two simple procedures for quantifying macronutrient‐specific habitat conditions without requiring information about the natural prey. Both procedures assume that predators forage for nutrients rather than specific prey. Our results show that 10 of 11 species were food limited. Five species were lipid limited and one species was protein limited in the field. Co‐existing predator species showed considerable segregation of fundamental macronutritional niches. A linear relationship between specific nutrient limitation and the target lipid:protein (L:P) intake ratio indicates that species with high L:P target are more protein limited while species with low L:P target are more lipid limited. The study illustrates how species within a natural assemblage vary in nutritional niche and in specific nutrient limitation.     

Evidence for the presence of sexual pheromones in free-spawning starfish

Filtered natural seawater passed through a tank containing ripe female starfish of the forcipulate species Asterias forbesi (Desor 1848) and Orthasterias koehleri (de Loriol 1897) contains low concentrations of a substance which attracts the sperm of these species. Release of this substance is sex specific in at least one species, O. koehleri. Immature females apparently do not release the sperm attractant. Daily testing for sperm attractant in seawater in the Friday Harbor Laboratories' aquarium system, Washington, U.S.A., in the spring and summer of 1986 indicates that incursions of sperm attractant-bearing water occur frequently during the starfish reproductive season. Testing of seawater taken directly from the San Juan Channel in the summer of 1987 reveals that sperm attractant is also present at irregular intervals in natural seawater. There is a definite correlation between the sperm attractant incursions in the laboratory seawater and initiation of spawning of starfish in the laboratory. The periodic presence of a long-lived bio-active sex-specific substance in natural seawater during the reproductive season of starfish suggests that benthic invertebrates may communicate sexual identity and readiness to spawn by means of waterborne pheromones.     

From natural woodlands to cultivated land: diversity of fruit‐feeding butterflies and beetles in the mid‐Zambezi valley,northern Zimbabwe

We tested the effect of cultivation on butterfly (Nymphalidae: Charaxes) and beetle (Coleoptera: Scarabaeidae: Cetoniinae) species richness and abundance along a cultivation intensification gradient. Results showed significant differences in species richness and abundance between natural woodlands and cultivated landscapes with larger differences in areas of high cultivation intensity. The results indicate that natural woodland clearing for cultivation purposes has negative impacts on arthropod diversity, a situation more severe in highly intensified cultivated areas. Our results imply that mosaics of different land‐use units, each in a different phase of clearance‐cultivation‐abandonment‐recovery‐clearance cycle could counter the negative effects of cultivation intensity on arthropod diversity.     

Reduced root anchorage of freshwater plants in sandy sediments enriched with fine organic matter

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Artificial Seed Production and Cultivation of the Edible Brown Alga, Sargassum Fulvellum (Turner) C. Agardh: Developing a new Species for Seaweed Cultivation in Korea

Sargassum fulvellum is a brown alga recently introduced to the seaweed cultivation industry in Korea. There is current interest in the commercial scale of aquaculture of this species. For the artificial seeding and cultivation of this alga, growth and maturation were investigated from September 2002 to August 2003. Indoor culture experiments for maturation induction were also conducted at temperatures of 5, 10, 15, 20 and 25 ^∘C and irradiances of 20, 50, 80 and 100 μmol photons m⁻² s⁻¹ under 16:8 h (L:D) photoperiod. Within a given culture test range, higher temperature and irradiance levels favoured the maturation of receptacles in S. fulvellum. Using temperature and irradiance control for thalli, artificial seed production of this species could be done one month earlier than thalli matured in nature. Under natural condition, receptacle formation of the plants began in February, and the eggs were released from March to April. For mature thalli of 200 g wet wt., artificial seeding was complete enough for attachment on seed strings of 100 m. Mean production obtained from the artificial seeding technique in situ was 3.0 kg wet wt m⁻¹ of culture rope during the cultivation period.     

搅拌式光生物反应器培养海带配子体细胞：不同脉冲补料方式的研究

本文以批次培养为对照,研究了七种脉冲补料方式对搅拌式光生物反应器中培养大型褐藻海带配子体细胞生长和培养液内氮磷营养盐消耗的影响,并首次探讨了脉冲补料方式下不同补料时间点和补料量的影响作用。培养条件设定为50 mg DCW (细胞干重) L-1接种密度、培养液为改良的APSW人工海水、光强60 µE m-2 s-1、光周期16/8 h L/D、通气速率和搅拌速率分别为50 mL min-1和100 rpm。结果表明少量补料利于细胞对氮磷的协同吸收,进而利于生物量扩增。当培养液内氮磷富足或耗尽时补料对于生物量大量生产效果甚微,可能由于氮磷吸收变缓、其储存现象显著,或是其吸收协同性降低。文中当细胞生长至对数中期开始频繁补加少量氮磷营养盐,即维持培养液内氮磷浓度在各自起始浓度的1/3至1/2之间,对生物量生产最有效,生物量增长倍数高达12.270倍。     

SELENIUM: AN ESSENTIAL ELEMENT FOR GROWTH OF THE COASTAL MARINE DIATOM THALASSIOSIRA PSEUDONANA (BACILLARIOPHYCEAE)1,2

An obligate requirement for selenium is demonstrated in axenic culture of the coastal marine diatom Thalassiosira pseudonana (clone 3H) (Hust.) Hasle and Heimdal grown in artificial seawater medium. Selenium deficiency was characterized by a reduction in growth rate and eventually by a cessation of cell division. The addition of 10⁻¹⁰ M Na₂SeO₃ to nutrient enriched artificial seawater resulted in excellent growth of T. pseudonana and only a slight inhibition of growth occurred at Na₂SeO₃ concentrations of 10⁻³ and 10⁻² M. By contrast, Na₂SeO₄ failed to support growth of T. pseudonana when supplied at concentrations less than 10⁻⁷ M and the growth rate at this concentration was only one quarter of the maximum growth rate. The addition of 10⁻³ and 10⁻² M Na₂SeO₄ to the culture medium was toxic and cell growth was completely inhibited. Eleven trace elements were tested for their ability to replace the selenium requirement by this alga find all were without effect. In selenium-deficient and selenium-starved cultures of T. pseudonana cell volume increased as much as 10-fold as a result of an increase in cell length (along the pervalvar axis) but cell width was constant. It is concluded that selenium is an indispensable element for the growth of T. pseudonana and it should be included as a nutrient enrichment to artificial seawater medium when culturing this alga.     

SELENIUM: AN ESSENTIAL ELEMENT FOR GROWTH OF THE COASTAL MARINE DIATOM THALASSIOSIRA PSEUDONANA (BACILLARIOPHYCEAE)1,2

An obligate requirement for selenium is demonstrated in axenic culture of the coastal marine diatom Thalassiosira pseudonana (clone 3H) (Hust.) Hasle and Heimdal grown in artificial seawater medium. Selenium deficiency was characterized by a reduction in growth rate and eventually by a cessation of cell division. The addition of 10⁻¹⁰ M Na₂eO₃ to nutrient enriched artifical seawater resulted in excellent growth of T. pseudonana and only a slight inhibition of growth occurred at Na₂SeO₃ concentrations of 10⁻³ and 10_-2 M. By contrast, Na₂SeO₄ failed to support growth of T. pseudonana when supplied at concentrations less than 10⁻⁷ M and the growth rate at this concentration was only one quarter of the maximum growth rate. The addition of 10⁻³ and 10⁻² M Na₂SeO₄ to the culture medium was toxic and cell growth was completely inhibited. Eleven trace elements were tested for their ability to replace the selenium requirement by this alga and all were without effect. In selenium-deficient and selenium-starved cultures of T. pseudonana cell volume increased as much as 10-fold as a result of an increase in cell length (along the pervalvar axis) but cell width was constant. It is concluded that selenium is an indispensable element for the growth of T. pseudonana and it should be included as a nutrient enrichment to artificial seawater medium when culturing this alga.