Susceptibility to oxidative stress of the Mediterranean demosponge Petrosia ficiformis : role of endosymbionts and solar irradiance

 The effects of elevated pO₂ and irradiance as inducers of prooxidant conditions have been investigated in the Mediterranean demosponge Petrosia ficiformis (Poiret, 1789). This species lives symbiotically with the autotrophic cyanobacterium Aphanocapsa feldmanni, the abundance of which is controlled by the intensity of light irradiance. In the presence of symbionts, tissues of P. ficiformis were characterized by a general enhancement of antioxidant defenses as compared to aposymbiotic specimens. The main differences included higher activities of several antioxidant enzymes and a greater capability to neutralize various forms of oxyradicals, as indicated by the total oxyradical scavenging capacity (TOSC) assay. Elevated pO₂, more than light, appeared to be the primary factor inducing prooxidant pressure in the Mediterranean sponge; in fact, irrespective of the solar irradiance experienced by the sponge, symbiotic specimens showed comparable activities of antioxidant enzymes and a similar scavenging capacity towards various reactive oxygen species. However, the potential toxicity of photodynamic production of reactive oxygen species was demonstrated in organisms from more irradiated sites, as the levels of antioxidant defenses were lowered in the outer layer of the sponge. The role of enhanced antioxidant defenses in protecting symbiotic specimens, also from oxyradical-mediated toxicity of light exposure, was supported by translocation experiments; aposymbiotic sponges did not survive when moved to conditions of elevated solar irradiance, while no effects were observed in symbiotic specimens if translocated and/or deprived of symbionts. Received: 23 November 1999 / Accepted: 13 June 2000     

Host specificity of the symbiotic cyanobacterium<Emphasis Type="Italic"> Oscillatoria spongeliae</Emphasis> in marine sponges,<Emphasis Type="Italic"> Dysidea</Emphasis> spp.

Marine sponges can host a variety of cyanobacterial and bacterial symbionts, but it is often unclear whether these symbionts are generalists that occur in many host species or specialists that occur only in certain species or populations of sponges. The filamentous cyanobacterium Oscillatoria spongeliae is found in the sponges Dysidea n. sp. aff. herbacea 1A and 1B, and similar cyanobacteria are found in D. n. sp. aff. granulosa. We amplified and sequenced sponge nuclear ribosomal DNA (rDNA) and cyanobacterial 16S rDNA from specimens of these three sponges. We then used these sequences to construct phylogenies for host sponges and their symbiotic cyanobacteria. Each of these three sponge species hosts a distinct cyanobacterial clade, suggesting a high degree of host specificity and potential coevolution between symbiotic cyanobacteria and their host sponges.     

A brominated secondary metabolite synthesized by the cyanobacterial symbiont of a marine sponge and accumulation of the crystalline metabolite in the sponge tissue

The dictyoceratid marine sponge Dysidea herbacea (Keller, 1889) is common in shallow waters of the tropical Pacific Ocean. Polybrominated biphenyl ethers such as 2-(2,4-dibromophenyl)-4,6-dibromophenol (1) are characteristic secondary metabolites of some specimens of this sponge and may represent as much as 12% of the dry weight. We have found 1 to be deposited as conspicuous crystals throughout the sponge tissue. The dominant prokaryotic endosymbiont in the mesohyl of the sponge is a filamentous cyanobacterium (Oscillatoria spongeliae), although a vacuole-containing, heterotrophic bacterium is also present. The cyanobacteria were separated from the sponge cells and heterotrophic bacteria by flow cytometry. Coupled gas chromatography—mass spectrometry and proton nuclear magnetic-resonance spectroscopy revealed that the major brominated Compound 1 isolated from the intact symbiotic association is found in the cyanobacteria and not in the sponge cells or heterotrophic bacteria. This suggests that the production of the compound is due to the cyanobacterium, and not to the sponge or symbiotic heterotrophic bacteria, as had been suggested earlier.     

Sponge-specific bacterial symbionts in the Caribbean sponge, Chondrilla nucula (Demospongiae, Chondrosida)

Marine sponges harbor dense and highly diverse bacterial communities, and some percentage of the microflora appears to be specialized for the sponge habitat. Bacterial diversity was examined in Chondrilla nucula Schmidt to test the hypothesis that some subset of sponge symbiont communities is highly similar regardless of the species of host or habitat requirements of the host. C. nucula was collected from a mangrove channel on Lower Matcumbe Key in the Florida Keys (25°53′N; 80°42′W) in August 1999. Domain-specific universal bacterial primers were used to amplify the 16S rDNA gene from genomic DNA that had been extracted from sponges and the surrounding water. An RFLP technique was used to assess diversity of sponge-associated and environmental bacterial communities. The clone library from C. nucula contained 21 operational taxonomic units (OTUs). None of the 53 OTUs from adjacent water samples were found in the C. nucula library indicating that a distinct community was present in the sponge. Sequence analysis indicated that C. nucula harbors a microbial community as diverse as the microbes from other sponges in different habitats around the world. Phylogenetic analysis placed several C. nucula clones in clades dominated by bacteria that appear to be sponge specialists (e.g., Acidobacteria, Bacteroidetes, and Cyanobacteria). Proportional representation of major bacterial taxonomic groups represented in symbiont communities was compared as a function of geographic location of sponge hosts. This study supports the hypothesis that sponges from different oceans existing in dissimilar habitats harbor closely related bacteria that are distinct from other bacterial lineages and appear specialized for residing within sponges.     

Cortical and endosomal structure of the marine sponge Stelletta grubii

The fine structure of the marine astrophorid sponge Stelletta grubii is described for the first time. The following new data are presented: spongin is present, choanocyte chambers are diplodal, intercellular symbiotic bacteria are numerous and unequally distributed in the cortex and endosome, and collagenous fibril bundles are associated with lophocyte activity and are not elastic fibers. The cortex contains numerous fibril bundles, fewer symbionts, very few cells, and transitional zones with higher archeocyte density near the surface and endosome. Limited phagocytosis of the bacterial symbionts is observed. This species appears to be dioecious and oviparous. These observations suggest that the enigmatic species Chondrosia reniformis is closely related to S. grubii and that it should be placed within or near the astrophorids. The rhizoids of the red alga Phyllophora palmettoides penetrate the sponge tissue without eliciting the development of a structurally specialized contact zone in the sponge matrix or of a limiting epithelium.     

Microbial associations in sponges. II. Numerical analysis of sponge and water bacterial populations

Three taxonomically distant sponges Pericharax heteroraphis, Jaspis stellifera and Neofibularia irata contain phenotypically similar bacterial symbionts which differ from bacteria in the ambient water. These symbionts are predominant in the sponges and were detected after computer analysis of 526 heterotrophic bacterial strains tested for 76 characters. These facultative anaerobic symbionts metabolize a wide range of compounds and may be important in removing waste products while the sponges are not circulating water. The bacteria produce sticky-mucoid colonies and thus would contribute to sponge structural rigidity. The fourth sponge Ircinia wistarii contains a mixed aerobic population similar to that in the ambient water. The majority of the bacteria are located around the inhalant canals, facilitating the uptake of dissolved organic matter and oxygen from the incoming water.     

The effects of symbiotic state on heterotrophic feeding in the temperate sea anemone <Emphasis Type="Italic">Anthopleura elegantissima</Emphasis>

The temperate sea anemone Anthopleura elegantissima is facultatively symbiotic with unicellular algae. Symbiotic A. elegantissima can supplement heterotrophic feeding with excess photosynthate from their algal partners, while asymbiotic individuals must rely solely on heterotrophy. A. elegantissima individuals were collected from Swirl Rocks, Washington (48°25′6″ N, 122°50′58″ W) in July 2010, and prey capture and feeding characteristics were measured to determine whether asymbiotic individuals are more efficient predators. Feeding abilities were then measured again after a 3-week exposure to full sunlight or shaded conditions. Freshly collected asymbiotic anemones had larger nematocysts, but symbiotic individuals showed greater nematocyte sensitivity. Sunlight enhanced digestion and reduced cnida density in all anemones regardless of symbiotic state. Results suggest that the phototropic potential of A. elegantissima, as influenced by symbiotic condition, has little effect on heterotrophic capacity. The anemones appear to maximize heterotrophic energy input independent of the presence or identity of their algal symbionts.     

Isolation of Oscillatoria spongeliae,the filamentous cyanobacterial symbiont of the marine sponge Dysidea herbacea

The tropical marine sponge Dysidea herbacea (Keller) (Dictyoceratidae: Dysideidae) is always found associated with the filamentous cyanobacterium (blue-green alga) Oscillatoria spongeliae (Schulze) Hauck (Cyanophyceae: Oscillatoriaceae), which occurs abundantly throughout the sponge mesohyl. Intact, metabolically active, trichomes of O. spongeliae were isolated from the sponge by chopping the sponge tissue with a razor blade and squeezing the trichomes into a seawater-based medium containing polyvinylpyrrolidone, bovine serum albumin, dithiothreitol, glycerol, KCl and Na₂CO₃. The isolated cyanobacteria were concentrated by centrifugation and then washed several times in fresh medium. The isolated O. spongeliae have photosynthetic rates which are similar to the intact sponge-alga association for periods of at least 6 h after isolation. Addition of sponge homogenate to the isolated cyanobacteria causes rapid cell lysis.     

Effect of iron and dissolved silica on primmorphs of Petrosia ficiformis (Poiret, 1789)

The suitability of the primmorphs system as a good model for biotechnological applications led researchers on primmorphs to look for a medium to stimulate cell proliferation and therefore growth of aggregates. Recent efforts have focused on the use of Fe⁺³ and Si that, supplemented to cell culture medium, were found to be promising for growth and morphogenesis of the sponge Suberites domuncula. In this work, we analysed the effect of iron and dissolved silica on primmorphs of Petrosia ficiformis, by testing them at different concentrations in successive experiments. The purpose of these experiments was to test their effect on primmorphs and individuate their optimal concentration for this species. Our results suggest a negative effect of iron on primmorphs of P. ficiformis and a positive effect of silica on primmorphs size and spiculogenesis at a concentration of 120 μM.     

Symbiotic zooxanthellae enhance boring and growth rates of the tropical sponge Anthosigmella varians forma varians

Several species of boring sponges harbor symbiotic zooxanthellae, and it is believed that the symbiont enhances boring activity of host sponges. This hypothesis was tested using manipulative field experiments to assess the effect of intracellular zooxanthella populations on boring rates of the tropical sponge Anthosigmella varians forma varians. Portions of sponge were attached to 60 calcium carbonate blocks of known weight. Three sets of 10 blocks were grown at high light levels and three sets of 10 blocks were grown at low light levels for 105 d in the Florida Keys, Florida, USA. Boring rates, growth rates (lateral growth and within-substratum tissue penetration), and zooxanthella populations were measured at the end of the experiment. Absolute rates of boring and growth of A. varians forma varians were significantly greater when zooxanthella densities were higher. Boring rate and tissue penetration related to final surface area of sponge attachment was also enhanced when zooxanthella densities were higher, suggesting that the symbiont plays a physiological role in the decalcification process. This is in contrast to the role that zooxanthellae play in coral hosts. Based on the results of this study, it appears that the presence of zooxanthellar symbionts has important ecological and life-history consequences for host sponges. Ability to laterally overgrow competitors will be correlated with the size and activity of zooxanthella populations. In addition, the fitness of host sponges will be enhanced by algal symbionts, since greater penetration within substrata will result in an increase in production of tissue that can be converted into storage, feeding and reproductive functions.     

The relative contribution of dinoflagellate photosynthesis and stored lipids to the survivorship of symbiotic larvae of the reef-building corals

The long-distance dispersal of larvae provides important linkages between populations of reef-building corals and is a critical part of coral biology. Some coral planulae have symbiotic dinoflagellates (Symbiodinium spp.) that probably provide energy in addition to the lipids provisioned within the egg. However, our understanding of the influence of symbionts on the energy metabolism and survivorship of planulae remains limited. This study examines the relative roles of symbiotic dinoflagellate photosynthesis and stored lipid content in the survivorship of the developing stages of the corals Pocillopora damicornis and Montipora digitata. We found that survivorship decreased under dark conditions (i.e. no photosynthetic activity) for P. damicornis and M. digitata at 31 and 22 days after release/spawning, respectively. The lipid content of P. damicornis and M. digitata planulae showed a significant decrease, at a higher rate, under dark conditions, when compared with light conditions. When converted to energy equivalents, the available energy provided by the depletion of lipids could account for 41.9 and 84.7% of larval metabolism for P. damicornis (by day 31) and 38.4 and 90.1% for M. digitata (by day 21) under light and dark conditions, respectively. This finding indicates that not all energy requirements of the larvae are met by lipids: energy is also sourced from the photosynthetic activities of the symbiotic dinoflagellates within these larvae, especially under light conditions. In addition, the amounts of three main lipid classes (wax esters, triglycerides, and phospholipids) decreased throughout the experiment in the planulae of both species, with the wax ester content decreasing more rapidly under dark conditions than under light conditions. The observations that the planulae of both species derive considerable amounts of energy from wax esters, and that symbiotic dinoflagellates enable larvae to use their stores at lower rates, suggested that symbiotic dinoflagellates have the potential to extend larval life under light conditions.     

Transmission,plasticity and the molecular identification of cyanobacterial symbionts in the Red Sea sponge <Emphasis Type="Italic">Diacarnus erythraenus</Emphasis>

Cyanobacterial symbionts in the sponge Diacarnus erythraenus from the Red Sea were identified in both adult sponges and their larvae by 16S rDNA sequencing. A single cyanobacterial type was found in all samples. This cyanobacterial type is closely related to other sponge cyanobacterial symbionts. The cyanobacterial rDNA, together with the morphological analysis by electron and fluorescence microscopy, provided evidence for vertical transmission of the symbionts in this sponge. In addition, we show phenotypic plasticity of the symbionts inside the sponge, probably as a result of variability in light availability inside the sponge tissue. Finally, the reproduction of Diacarnus erythraenus is also described.Matan Oren, Laura Steindler have contributed equally to the work.     

Onset of symbiosis and distribution patterns of symbiotic dinoflagellates in the larvae of scleractinian corals

The establishment of symbiosis in early developmental stages is important for reef-building corals because of the need for photosynthetically derived nutrition. Corals spawn eggs and sperm, or brood planula larvae and shed them into the water. Some coral eggs or planulae directly inherit symbiotic dinoflagellates (Symbiodinium spp.) from their parents, while others acquire them at each generation. In most species examined to date, the larvae without dinoflagellates (aposymbiotic larvae) can acquire symbionts during the larval stage, but little is known regarding the timing and detailed process of the onset of symbiosis. We examined larval uptake of symbiotic dinoflagellates in nine species of scleractinian corals, the onset of symbiosis through the early larval stages, and the distribution pattern of symbionts within the larval host, while living and with histology, of two acroporid corals under laboratory conditions. The larvae acquired symbiotic dinoflagellates during the planktonic phase in all corals examined which included Acropora digitifera, A. florida, A. intermedia, A. tenuis, Isopora palifera, Favia pallida, F. lizardensis, Pseudosiderastrea tayamai, and Ctenactis echinata. The larvae of A. digitifera and A. tenuis first acquired symbionts 6 and 5 days after fertilization, respectively. In A. digitifera larvae, this coincided with the formation of an oral pore and coelenteron. The number of symbiotic dinoflagellates increased over the experimental periods in both species. To test the hypothesis that nutrients promotes symbiotic uptake, the number of incorporated dinoflagellates was compared in the presence and absence of homogenized Artemia sp. A likelihood ratio test assuming a log-linear model indicated that Artemia sp. had a significantly positive effect on symbiont acquisition. These results suggest that the acquisition of symbiotic dinoflagellates during larval stages is in common with many coral species, and that the development of both a mouth and coelenteron play important roles in symbiont acquisition.     

Ultrastructural aspects of the symbiosis between two species of the genus Aphanocapsa (Cyanophyceae) and Ircinia variabilis (Demospongiae)

The association between two species of the genus Aphanocapsa (Cyanophyceae) and the sponge Ircinia variabilis has been studied by electron microscopy. A. feldmanni is localized in the mesohyl or inside the cells of the sponge, while the larger A. raspaigellae is located only in an extracellular position inside cavities of the mesohyl. Both algae differ from other symbiotic Cyanophyceae in having a normal cell wall. They are able to reproduce in symbiotic condition, but also undergo, in their various extracellular and intracellular positions, a massive process of disintegration. A large amount of algal material is dispersed in the sponge tissues, which is a confirmation, at the ultrastructural level, of trophic relationships in the symbiosis Aphanocapsa-Ircinia.     

Bacterial symbionts as an additional cytological marker for identification of sponges without a skeleton

Symbiotic bacteria from six Oscarella species (adults and embryos) collected in the Mediterranean Sea (O. lobularis, O. tuberculata, O. imperialis, O. microlobata, O. viridis) and the Sea of Japan (O. malakhovi) were investigated by scanning electron microscopy and transmission electron microscopy. In most cases, symbionts are rather numerous. Each sponge species has a definite set of bacterial morphological types. All bacteria are extracellular. Symbionts occupy the mesohyl of adult sponges or intercellular space in embryos and are often in contact with mesohylar filaments or cells. Bacteria of some morphotypes have characteristic blebs. Most symbionts are gram-negative, and two types of bacteria have traits of Archaea and one type of bacteria is similar to Planctomycetes. Data on morphology of bacterial symbionts can be a good additional character for identification of Oscarella species, which have no skeleton.     

Effects of starvation,and light and dark on the energy metabolism of symbiotic and aposymbiotic sea anemones,Anthopleura elegantissima

Rates of oxygen and carbon-dioxide exhange were measured in symbiotic and aposymbiotic specimens of the sea anemone Anthopleura elegantissima while fed and starved under light or dark conditions. Respiratory quotients indicated that fed anemones switched from a carbohydrate to a fat catabolism when starved, with the exception that symbiotic individuals starved in the light showed a pronounced carbohydrate catabolism for over 1 month. The source of the carbohydrate was probably photosynthate translocated by the dinoflagellate Symbiodinium (=Gymnodinium) microadriaticum (Freudenthal) living in the anemones' tissues. The starved symbiotic anemones maintained in the light had lipid levels not significantly different from fed controls and 44 to 61% higher than starved aposymbiotic anemones after 1 month. Thus, the quality and quantity of the metabolic flux from the symbionts to the sea anemone were sufficient to conserve the host's lipid reserves.     

The importance of methane and thiosulfate in the metabolism of the bacterial symbionts of two deep-sea mussels

Undescribed hydrocarbon-seep mussels were collected from the Louisiana Slope, Gulf of Mexico, during March 1986, and the ultrastructure of their gills was examined and compared to Bathymodiolus thermophilus, a mussel collected from the deep-sea hydrothermal vents on the Galápagos Rift in March 1985. These closely related mytilids both contain abundant symbiotic bacteria in their gills. However, the bacteria from the two species are distinctly different in both morphology and biochemistry, and are housed differently within the gills of the two mussels. The symbionts from the seep mussel are larger than the symbionts from B. thermophilus and, unlike the latter, contain stacked intracytoplasmic membranes. In the seep mussel three or fewer symbionts appear to be contained in each host-cell vacuole, while in B. thermophilus there are often more than twenty bacteria visible in a single section through a vacuole. The methanotrophic nature of the seep-mussel symbionts was confirmed in ¹⁴C-methane uptake experiments by the appearance of label in both CO₂ and acid-stable, non-volatile, organic compounds after a 3 h incubation of isolated gill tissue. Furthermore, methane consumption was correlated with methanol dehydrogenase activity in isolated gill tissue. Activity of ribulose-1,5-biphosphate (RuBP) carboxylase and ¹⁴CO₂ assimilation studies indicate the presence of either a second type of symbiont or contaminating bacteria on the gills of freshly captured seep mussels. A reevaluation of the nutrition of the symbionts in B. thermophilus indicates that while the major symbiont is not a methanotroph, its status as a sulfur-oxidizing chemoautotroph, as has been suggested previously, is far from proven.     

Variability in the chemical defense of the sponge Chondrilla nucula against predatory reef fishes

Chondrilla nucula is a common Caribbean demosponge that grows in a range of habitats, from coral reefs to mangrove swamps. On reefs, C. nucula grows as a thinly encrusting sheet, while in mangrove habitats it surrounds submerged mangrove roots as fleshy, lobate clumps. Previous feeding experiments using predatory reef fish revealed a high degree of variability in the chemical defenses of C. nucula. The present study was undertaken to determine whether a relationship exists between habitat, growth form, and chemical defense of C. nucula. Both laboratory and field feeding-assays of crude extracts confirmed that C. nucula possesses a chemical defense with high intercolony variability, but there was no significant variation in feeding deterrency between reef and mangrove habitats at either geographic location (Bahamas and Florida). Extracts of C. nucula collected during September and October 1994 from the Bahamas were significantly more deterrent than those collected during August 1993, May 1994, and May 1995 from Florida, and extracts of these spring and summer Florida collections were more deterrent than extracts of C. nucula collected in December 1994 and February 1995 in the same locations. There was no evidence that deterrent compounds were concentrated in the surface tissues of the sponge, or that chemical defense could be induced by simulated predation. Laboratory and field assays of the fractionated crude extract revealed that feeding deterrency was confined to the most polar metabolites in the extract. Field transplants were used to determine whether predation influenced the growth form of C. nucula. Uncaged sponges transplanted from the mangrove to the reef were readily consumed by spongivorous reef fishes. Lobate mangrove sponges became thinner after being caged on the reef for 3 mo, but encrusting reef sponges did not become thicker after being caged in the mangroves for the same period of time. Reef sponges that were caged for 3 to 15 mo thickened by only a small amount (<1 mm) compared to uncaged and open-caged (i.e. in cages lacking tops) sponges. Simulated bite marks on both reef and mangrove sponges were repaired at a rapid rate (0.8 to 1.6 mm d⁻¹). Fish predation has an important impact on the distribution and abundance of C. nucula, but the thin growth form common to reef environments may be more the result of hydrodynamics than of grazing by spongivorous fishes. Received: 6 October 1997 / Accepted: 19 March 1998     

Heterotrophic nutrition of the marine pennate diatom Nitzschia angularis var. affinis

The nutritional pattern for heterotrophic growth of Nitzschia angularis var. affinis (Grun.) Perag. is more complex than for other diatom species studied previously. This species grew slowly in the dark in the presence of single amino acids, either glutamate or alanine; other amino acids when supplied singly were not used as substrates. Carbon from glutamate was converted to cell carbon with an efficiency of 43%. Glutamine was inhibitory both in the light and in the dark, and aspartate inhibited heterotrophic growth on glutamate. Glucose and tryptone supplied singly did not support heterotrophic growth, but when combined, together they allowed for rapid growth of N. angularis (generation time of 16 h). Glucose in combination with glutamate, alanine, aspartate, or asparagine (but not with any other amino acids) also supported growth in the dark, at a rate considerably more rapid than with glutamate alone. In the presence of excess glucose and limiting concentrations of glutamate, approximately 50% of the cell carbon for heterotrophic growth came from glucose, while in combination with tryptone about 25% of the cell carbon came from glucose. Amino acids were taken up by cells grown either photoautrophically or in the dark in the presence or absence of organic substrates; uptake rates were some-what higher for dark-grown than for light-grown cells. Glucose was taken up only by dark-grown cells; induction of a glucose uptake system in the dark required the presence of glutamate but not of glucose. The rates of uptake of glutamate and glucose by cells incubated in the dark with glutamate were sufficiently high to account for the observed rates of growth on these substrates in the dark. The uptake systems of N. angularis have relatively high affinities for glucose (K _s =0.03 mM) and glutamate (K _s =0.02 mM).Contribution No. 890 from the Department of Oceanography, University of Washington, Seattle, Washington 98195, USA.     

Utilization of glutamate and glucose for heterotrophic growth by the marine pennate diatom Nitzschia laevis

Nitzschia laevis Hustedt grew in the dark in the presence of either glutamate or glucose as substrate. Complex mixtures of yeast extract or tryptone plus lactate also supported good heterotrophic growth, while tryptone alone only supported very slow growth in the dark. The observed growth rates of N. laevis in the dark at different concentrations of glutamate or glucose could be accounted for by the measured uptake rates of these compounds. The affinity of the uptake systems for glutamate and glucose (K _s =0.03 mM for each) was quite high, and similar for dark- and light-grown cells. The lack of a lag-phase when cells were transferred from photoautotrophic to heterotrophic growth conditions can be explained by the presence of uptake systems for glutamate and glucose in ligh--grown cells, as well as in dark-grown cells. However, the uptake capacity was generally higher in the latter than the former. N. laevis also took up alanine and lactate according to Michaelis-Menten kinetics, with a K _s for alanine of 0.02 mM and for lactate of 0.4 mM. Malate and glycerol were not taken up to a significant extent by the cells. Cells grown in continous light had a doubling time of 18 h. The shortest doubling time observed in the dark on glutamate was 48 h and on glucose 24 h. Glutamate was used for heterotrophic growth with an efficiency of 43% and glucose with an efficiency of 48%.Contribution No., 945 from the Department of Oceanography, University of Washington, Seattle, Washington 98195, USA.