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.     

A NEW,ARTIFICIAL SEA WATER THAT FACILITATES GROWTH OF LARGE NUMBERS OF CELLS OF ACETABULARIA ACETABULUM (CHLOROPHYTA) AND REDUCES THE LABOR INHERENT IN CELL CULTURE1

We designed a new, artificial seawater (Ace 25) in order to grow bulk cultures of Acetabularia acetabulum (L.) Silva with a minimum of labor. To this end, we modified the traditional recipe for cell growth (Müller's medium as modified by Schweiger et al.) We eliminated five of the inorganic chemicals and determined the optimum concentration for 16 of the remaining 18 inorganic chemicals from modified Müller's seawater. Ace 25 enables growth of A. acetabulum from the beginning of the juvenile phase through gametangial formation in 11 weeks at high cell densities without medium replenishment. This represents a 98% reduction in the seawater volume required to mature each cell, a 30–40% reduction of the duration of the life cycle, an estimated 80% reduction in labor, and a 50–95% reduction in the space required for culturing A. acetabulum as compared with traditional procedures. These improvements may facilitate studies that require large numbers of cells such as population studies, genetics, and biochemistry, contribute to understanding the nutritional requirements of marine algae, and extend the use of this cell to those who lack the space or manpower to grow the cells in the traditional manner.     

INFLUENCE OF SALINITY ON THE FORM OF ASTEROCYTIS IN PURE CULTURE1

The growth form of an isolate of Asterocytis ornata in pure culture changed from filamentous in a full-strength seawater medium to a unicellular or bicellular form closely resembling the genus Chroothece in a medium of quarter-strength seawater. The alga did not grow in media of lower salinities, or in media in which seawater was replaced by KCl, NaCl, or CaCl₂. The formation of filaments from single cells could not be attributed to osmolarity. The controlling factors seemed to be the concentrations of cations, notably Na⁺ and Mg⁺⁺. However, the filaments produced experimentally in a defined medium, were never as extensively developed as those occurring in seawater, an indication that further studies should be done. The occurrence of the Asterocytis form, in freshwater habitats remains to be explained. There may be distinct physiological races, if not true species, within the genus.     

Growth of Bacillus methanolicus in seawater-based media

Bacillus methanolicus has been proposed as a biocatalyst for the low cost production of commodity chemicals. The organism can use methanol as sole carbon and energy source, and it grows aerobically at elevated temperatures. Methanol can be made available from off-shore conversion of natural gas to methanol, through gas-to-liquid technology. Growth of the organism in seawater-based medium would further reduce the costs of chemical production performed near an off-shore natural gas source. The growth of strain PB1 (ATCC 51375) in shake flask experiments with trypticase soy broth medium showed minimal salt-inhibition at the concentration of NaCl in seawater. The ability of B. methanolicus PB1 to grow in Pacific Ocean water using methanol as a carbon and energy source was also tested. Following a simple adaptation procedure, PB1 was able to grow on methanol in semi-defined medium with 100% seawater with good growth yields and similar growth rates compared with those achieved on media prepared in deionized water.     

Screening of the culture media with different concentrations of nutrients for cultivation of the microalgae associated with the invertebrates of the White Sea

The growth and biomass accumulation of three microalgal strains of Desmodesmus (Scenedesmaceae, Chlorophyceae), 1Рm66В, 2Cl66E, and 3Dp86Е-1, isolated from the White Sea benthic invertebrates were studied under conditions of batch culture in different standard media (BG-11, Prat, Goldberg, Gromov, Tamiya, artificial seawater) and modified media. The culture condition, biomass accumulation, and uptake of nitrate and phosphate were recorded. A significant alkalization of the culture medium up to pH 10 has been observed during a vigorous growth of the microalgae. The most significant biomass accumulation has been recorded in BG-11 (in complete or modified medium with addition of artificial seawater), Tamiya, and Prat media. Addition of seawater did not affect the growth of Desmodesmus sp. in the nitrate-containing media, although that maintained growth of the microalgae in the nitrogen-lacking media without cell aggregation. The BG-11 medium appears suitable for isolation and cultivation of both symbiotic and free-living microalgae by all the tested features. The Prat medium is the best for maintaining the microalgal strains in living collection.     

Influence of aquatic microbiota on the survival in water of the human and eel pathogen Vibrio vulnificus serovar E

The eel and human pathogen Vibrio vulnificus serovar E (biotype 2) is seldom isolated from natural waters, although it can survive in sterilized artificial seawater microcosms for years. The main objective of the present study was to investigate whether aquatic microbiota can limit its survival and recovery from water samples. A set of preliminary experiments of survival in microcosms containing natural seawater and water from eel farms showed that the persistence of this pathogen was mainly controlled by grazing, and secondarily by bacterial competition. The bacterial competition was further analysed in artificial seawater microcosms co-inoculated with selected virulent serovar E (VSE) strains and potential competitors. Competitors included V. vulnificus biotype 1 isolates and strains of selected species that can grow on the selective media designed for V. vulnificus isolation from water samples. Evidences of bacterial competition that was detrimental for VSE recovery were recorded. Thus, some species produced a deleterious effect on VSE strains under starvation, and others were able to use the resources more efficiently under nutrient input. These results suggest that an overgrowth of more efficient competitor bacteria in conventional media used for isolation of V. vulnificus could mask the recovery of VSE strains and explain the scarcity of reports on the isolation of this human and eel pathogen from natural waters.     

Recirculating Culture for Chondracanthus Exasperatus

The red alga Chondracanthus exasperatus is a source of the phycocolloid carrageenan as well as an ingredient referred to as ‘intralamellar gel’ in a recently developed cosmetic formula (US Patent 6136 329). The high value of the cosmetic product has sparked renewed interest in cultivation of this species. Previous cultivation methods for this species include open water culture on nets and immersed cultivation in tanks supplied with flow‐through pumped seawater. The installation of a high capacity seawater supply, pumping and drain system is a major cost for flow through systems. Recirculating or re‐use seawater systems that minimize seawater turnover may offer significant cost savings over single‐pass, flow through seawater systems. In this research several options for minimizing seawater use have been tested: recirculating batch culture in which nutrient replenished (carbon dioxide and mineral nutrients) natural or artificial seawater is used with minimal turnover and spray culture in which plants are suspended in air saturated with nutrient replenished natural or artificial seawater medium. Small volume (<2 L), single‐plant bioreactors and larger multiplant, 20, 80 and 320 L (sea water volume) immersion and spray systems have been developed and tested. Results from these systems will be presented. Research supported by Washington Sea Grant, Washington Biotechnology Center and Soliv International Corporation.     

The only elements required by plants that are deficient in seawater are nitrogen,phosphorous and iron

It has been known for some time that mangroves grow in the intertidal areas of desert countries where fresh water flows into the sea, but this phenomenon has not been understood. In Eritrea we observed that mangroves grow where infrequent rains flow into the Red Sea. We theorized that the fresh water must bring elements needed for plant growth that are absent in seawater. We compared the composition of seawater to that of Zarrouk’s algae medium. All the elements in algae medium are in sufficient quantity in seawater except for nitrogen, phosphorous and iron. If we supply these elements we can grow any plant that can grow in seawater. We have also begun planting plants that can grow in sea water in the Sahara desert with sea water irrigation and fertilization with nitrogen, phosphorus and iron. We believe this will be major step in reducing hunger and poverty in the world.     

Short peptide induces an "uncultivable" microorganism to grow in vitro

Microorganisms comprise the bulk of biodiversity, but only a small fraction of this diversity grows on artificial media. This phenomenon was noticed almost a century ago, repeatedly confirmed, and termed the "great plate count anomaly." Advances in microbial cultivation improved microbial recovery but failed to explain why most microbial species do not grow in vitro. Here we show that at least some of such species can form domesticated variants capable of growth on artificial media. We also present evidence that small signaling molecules, such as short peptides, may be essential factors in initiating growth of nongrowing cells. We identified one 5-amino-acid peptide, LQPEV, that at 3.5 nM induces the otherwise "uncultivable" strain Psychrobacter sp. strain MSC33 to grow on standard media. This demonstrates that the restriction preventing microbial in vitro growth may be different from those offered to date to explain the "great plate count anomaly," such as deficiencies in nutrient composition and concentrations in standard media, medium toxicity, and inappropriate incubation time. Growth induction of MSC33 illustrates that some microorganisms do not grow in vitro because they are removed from their native communities and the signals produced therein. "Uncultivable" species represent the largest source of unexplored biodiversity, and provide remarkable opportunities for both basic and applied research. Access to cultures of some of these species should be possible through identification of the signaling compounds necessary for growth, their addition to standard medium formulations, and eventual domestication.     

Diatom artificial medium (DAM): a new artificial medium for the diatom <Emphasis Type="Italic">Haslea ostrearia</Emphasis> and other marine microalgae

Artificial media are used in physiological studies of microalgae to maintain consistent conditions from one experiment to another and these media must be adapted to the needs of the organism studied. The artificial medium, in this case named diatom artificial medium (DAM), was designed to maintain long-term cultures of Haslea ostrearia and 19 other planktonic microalgae, and to allow physiological studies related to metal metabolism. The biomass and biochemical composition of H. ostrearia grown in the DAM and in a modified Provasoli medium were compared to assess the suitability of this new artificial medium for the culture of this diatom. The DAM provided sufficient nutrients to allow H. ostrearia to grow as efficiently as in the enriched seawater medium, without negative impact on metabolism. The DAM was tested with 19 other microalgae in order to widen its potential use, and 18 of the 19 showed a good adaptation to this medium. The chemical speciation of metals (Cd, Cu, Pb, Zn) was assessed using a speciation mathematical model. The presence of EDTA resulted in the total complexation of the trace metals implying that they were present in a sole chemical species in the DAM.     

A SEAWATER MEDIUM FOR DINOFLAGELLATES AND THE NUTRITION OF CACHONINA NIEI1

Two media have been devised: an enriched seawater medium for culture of dinoflagellates and a defined medium for rapid growth of the dinoflagellate Cachonina niei. A wide range in salinity (10.23–42.38 g/liter NaCl) is tolerated by C. niei. Below 0.6 g/liter MgSO₄, 0.19 g/liter KCl, and 0.22 g/liter CaCl₂, the generation time greatly increases. Increase in MgSO₄ to 7.22 g/liter, KCl to 1.12 g/liter or CaCl₂ to 2.22 g/liter has little effect on generation time. The temperature optimum is 19–23 C. Saturating light intensity for growth is 1000 ft-c and for photosynthesis (determined manometrically) is slightly less than 2000 ft-c. Cachonina niei requires B₁₂ and thiamin. Neither silicate nor its competitive inhibitor germanate affects generation time or cell yield indicating silicon is not required. Of a variety of buffers tested, Tris is the best. Optimal growth occurs at pHs of 7.5–8.3. Glycerol is inhibitory and does not support dark growth.     

Short Peptide Induces an “Uncultivable” Microorganism To Grow In Vitro

Microorganisms comprise the bulk of biodiversity, but only a small fraction of this diversity grows on artificial media. This phenomenon was noticed almost a century ago, repeatedly confirmed, and termed the “great plate count anomaly.” Advances in microbial cultivation improved microbial recovery but failed to explain why most microbial species do not grow in vitro. Here we show that at least some of such species can form domesticated variants capable of growth on artificial media. We also present evidence that small signaling molecules, such as short peptides, may be essential factors in initiating growth of nongrowing cells. We identified one 5-amino-acid peptide, LQPEV, that at 3.5 nM induces the otherwise “uncultivable” strain Psychrobacter sp. strain MSC33 to grow on standard media. This demonstrates that the restriction preventing microbial in vitro growth may be different from those offered to date to explain the “great plate count anomaly,” such as deficiencies in nutrient composition and concentrations in standard media, medium toxicity, and inappropriate incubation time. Growth induction of MSC33 illustrates that some microorganisms do not grow in vitro because they are removed from their native communities and the signals produced therein. “Uncultivable” species represent the largest source of unexplored biodiversity, and provide remarkable opportunities for both basic and applied research. Access to cultures of some of these species should be possible through identification of the signaling compounds necessary for growth, their addition to standard medium formulations, and eventual domestication.     

龙须菜对海水氮磷富营养化的响应

龙须菜(Gracilaria lemaneiformis)是红藻门江蓠属大型海藻,已从山东青岛引种到广东省南澳岛等地栽培多年。深入了解其光合作用与环境条件的关系,对龙须菜的合理栽培及其在环境生态中的生物学作用和扩大其开发利用价值,具有重要的理论和现实意义。该文利用人为配置的不同程度N、P污染海水处理龙须菜,研究其生理生态特性对海水氮磷富营养化的响应。结果表明: 处理6 d后,龙须菜叶绿素a及3种藻胆蛋白含量和总抗氧化能力在轻度P(P 0.2 mg·L^-1)及重度N、P复合污染(P 10 mg·L^-1,N 55 mg·L^-1)情况下降低。叶绿素荧光参数的变化也表明上述处理对叶绿体PSⅡ造成不利影响,而在轻度N(N 0.9 mg·L^-1)和轻度N、P复合污染(P 0.2 mg·L^-1,N 0.9 mg·L^-1)海水中上述参数则变化不大,表现出龙须菜对轻度N及轻度N、P复合污染的耐受性。该实验条件下,轻度污染处理后,藻体中不因环境N、P浓度升高而富集N、P。表明龙须菜可以作为轻度富营养化海水水体的净化藻类。     

海水盐离子对耐盐粘球菌生长和发育的影响

黄褐粘球菌 (Myxococcusfulvus)HW 1菌株是分离自海水样品的耐盐粘细菌 ,能够在海水 (盐浓度为3.8% )或稀释海水条件下生长。随着生长环境中海水浓度的提高 ,HW 1在生长与分化发育上表现出不同于陆地粘细菌的特性。对海水中主要盐离子的进一步分析表明 ,Na+ 对HW 1生长有抑制作用 ,当Ca2 + 超过海水中浓度时对HW 1生长抑制最为明显 ,而Mg2 + 对HW 1生长几乎没有抑制作用 ,但能促进子实体的形成。海水盐成分中SO42 -,K+ 和不含K+ 的其它盐组合都能导致孢子抗热能力的提高。     

人工污水对秋茄幼苗形态及解剖构造的影响

在温室中建立模拟的秋茄湿地系统, 对照组用人工海水, 其余组分别用3 种不同浓度的人工污水定时定量对模拟系统进行灌溉, 持续1 年。结果表明污水影响植物根系的发育, 胚轴、茎和叶片的形态结构相应发生变化, 这些变化应该是秋茄幼苗为适应污染物而主动产生的, 因此其抗污性较强。     

Growth of Mycobacterium leprae under low oxygen tension

Despite numerous attempts, Mycobacterium leprae has yet to be cultivated in vitro. This organism has been considered as microaerophilic. The effects of various known gas mixtures on the in vitro growth of M. leprae were investigated. A gas mixture containing 2.5% O2 and 10% CO2 was found to be more favourable for the growth of this mycobacterium on artificial medium. Growth was evaluated by three parameters namely cell counts, bacterial ATP and DNA. An optimal growth of M. leprae, as determined by all three parameters, on both liquid and solid media was obtained between 18 and 24 weeks of incubation under optimal gas mixture. Solid medium which contained egg-yolk was relatively more beneficial for in vitro growth than the liquid medium. The cultivated bacilli exhibited some important characteristics specific for M. leprae, including growth in mouse foot-pads. The bacilli gradually lost their power of adaptation to grow on artificial media and did not show any ATP or DNA after about 36 weeks of incubation.     

Nitrapyrin-ammonium combination induces rapid multiplication of mixed cultures of the stalked bacterium Nevskia ramosa famintzin and other heterotrophic bacteria

Nevskia ramosa, a bacterium that is very difficult to grow in artificial culture media in its recognizable and natural habit was induced to grow and multiply rapidly in mixed culture in an amended ASM medium. ASM liquid medium, an inorganic mineral medium, amended with 0.5 g ml^-1 of nitrapyrin and various levels of ammonium chloride (25–100 M) produced very significant enrichment of Nevskia ramosa within 24 h, permitting formation of its characteristic bushy dichotomously branched, stalked colonies with the bacterial cells at the apices of branches. ASM medium amended with either nitrapyrin or ammonium singly failed to produce growth of N. ramosa. The nitrapyrinammonium combination in ASM medium also produced a significant increase in growth of other heterotrophic bacteria. Our results should be of value to bacteriologists interested in studying N. ramosa, a species which has received little attention thus far.     

Effect of eight growth media upon fermentation profiles of ten anaerobic bacteria

A study was made of the influence of eight growth media upon the fermentation end-product patterns obtained with ten species of anaerobes. Acidic and neutral end-products of fermentation were analysed by gas chromatography and the results were examined statistically by computer. Differences in end-product profile were at least as great between different media used to grow a single anaerobic species, as between different anaerobes grown in a single type of medium. When individual fermentation end-products produced by a single anaerobe species were examined, statistically significant differences were found between different media for individual end-products. It is concluded that standardisation of growth medium is essential in diagnostic laboratories concerned with identification of anaerobes with the aid of gas chromatography.     

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.     

Characteristics and Living Patterns of Marine Myxobacterial Isolates

The growth, morphology, and life cycle of two marine myxobacterial isolates, halotolerant Myxococcus fulvus strain HW-1 and halophilic Haliangium ochraceum strain SMP-2, were studied as models to determine the living patterns of myxobacteria in the ocean. The growth, morphology, and development of halotolerant strain HW-1 shifted in response to salinity. The optimal seawater concentration for growth of HW-1 was 0 to 80% (salinity, 0.1 to 2.9%), and the strain grew poorly in media with a salinity of more than 4%. The cells became shorter as the seawater concentration increased. The fruiting body structure was complete only on agar prepared with low concentrations of seawater or salts (less than 60% seawater; salinity, 2.1%), and rudimentary structures or even simple cell mounds appeared as the seawater concentration increased. In contrast, the halophilic strain SMP-2 was unable to grow without NaCl. The cell length and the morphology of the fruiting body-like structure did not change in response to salts. In seawater liquid medium, the cells of both strains were confirmed to be able to form myxospores directly from vegetative cells, but they could not do so in medium containing a low seawater concentration (10% or less). HW-1 cells from medium containing a high concentration of seawater grew independent of cell density, while cells from medium containing a low concentration of seawater (10% or less) showed density-dependent growth. SMP-2 cells showed density-dependent growth under all salinity conditions. The results suggest that the halotolerant myxobacteria are the result of degenerative adaptation of soil myxobacteria to the marine environment, while the halophilic myxobacteria form a different evolutionary group that is indigenous to the ocean.