ASCOPHYLLUM NODOSUM (PHAEOPHYTA) IN AXENIC CULTURE AND ITS RESPONSE TO THE ENDOPHYTIC FUNGUS MYCOSPHAERELLA ASCOPHYLLI AND EPIPHYTIC BACTERIA1

Ascophyllum nodosum (L.) Le Jol. was freed from bacteria and the endophytic fungus Mycosphaerella ascophylli Cotton by repeated treatment with chlorine solutions and grown in artificial seawater. Two types of axenic culture of different origin were obtained. Type 1 was developed from apices of A. nodosum collected in the sea. Type 2 was from plants which developed from adventitious embryos on rhizoids formed by type 1. This is the first time A. nodosum has been cultivated axenically. Growth of the axenic alga was increased by IAA, 21P and zeatin. Without external growth regulators some strains of the axenic alga deteriorated within a year; others developed a filamentous habit. Sulfur in a reduced state also stimulated growth. Addition of either glucose, mannose or mannitol to the medium caused the formation of calluslike layers of loosely packed colorless cells under the epidermis of the thalli and the epidermis was sloughed off. No increase in thallus length was noticed. Mycosphaerella ascophylli in axenic culture did not excude any substances stimulating growth of the alga, but that does not exclude an influence of the fungus on the alga in vivo. The fungus, when growing within the alga, seemed to have little influence on algal morphology. A bacterized but fungus-free A. nodosum was cultivated in an artificial seawater for 8 years. In the bacteria-free alga, the fungus protruded from the epidermis and evidently utilized the alga as a carbon source. The bacteria thus seem much more important than the fungus for normal growth of the Ascophyllum plant.     

The influence of culture conditions on mycorrhiza formation between the ectomycorrhizal fungus Pisolithus sp. and Afzelia africana Sm. seedlings

Interactions between an isolate of the ectomycorrhizal fungus Pisolithus sp. and Afzelia africana Sm. seedlings were studied at the structural and ultrastructural levels. Several different conditions were tested with or without sugar and in a sterile or nonsterile medium. In the growth cabinet, the A. africana/Pisolithus sp. interactions did not produce ectomycorrhizas. A fungal sheath was formed but no Hartig net, and an unusual host epidermal cell wall was observed. Hyphae of Pisolithus sp. induced modifications of epidermal cells of 15-day-old A. africana seedlings indicative of non-mycorrhizal interactions, such as wall thickening, wall ingrowth, papillae formation, degraded host wall material and the presence of intracellular hyphae. Wall ingrowth consisted of depositions of host cell wall materials giving a positive reaction for polysaccharides; however, wall thickenings and papillae showed no homogeneous reactions for polysaccharides. In glasshouse conditions, inocula of Pisolithus sp. in the form of spores or mycelia entrapped in peat-vermiculite added to sterilized soil produced typical ectomycorrhizae only with 6-month-old A. africana seedlings. Under these conditions, no conspicuous cell wall reactions occurred on A. africana roots. The results demonstrate that the establishment of an association between an ectomycorrhizal fungus and a potential host plant is strongly influenced by seedling age and/or environmental conditions. Therefore, in vitro synthesis is not a conclusive demonstration of a symbiotic relationship.     

A NEW HOMOTHALLIC VARIETY OF CHLAMYDOMONAS MOEWUSH (CHLOROPHYCEAE)1

A new homothallic variety of Chlamydomonas moewusii Gerloff var. monoica is described. When first isolated, the alga exhibited a very strong mating reaction (80–90% zygotes on 2 wk BBM agar slants), but after 2 mo in axenic culture, the reaction was significantly reduced in intensity. Attempts were made to restore the initial mating intensity by varying environmental conditions, but met with limited success. The alga did not grow heterotrophically in carbon-supplemented BBM medium.     

Nitrogen Effect on Water-Soluble Polysaccharide Accumulation in <Emphasis Type="Italic">Streblonema</Emphasis> sp. (Ectocarpales,Phaeophyceae)

The water-soluble polysaccharides of brown algae attract the increasing attention of researchers as an important class of polymeric materials of biotechnological interest. The sole source for production of these polysaccharides has been large brown seaweeds such as members of Laminariales and Fucales. A new source of water-soluble polysaccharides is suggested here: it is a filamentous brown alga Streblonema sp., which can be cultivated under controlled conditions in photobioreactors that allow obtaining algal biomass with reproducible content and quality of polysaccharides. The accumulation of water-soluble polysaccharides can be stimulated by macronutrient limitation. In response to nitrogen deficiency, Streblonema sp. accumulated water-soluble polysaccharides (WSPs) rich in laminaran. WSP accumulation started after 3–4 days following nitrate depletion and reached a plateau at around day 7. Polysaccharide accumulation was related to cellular nitrogen content. The critical internal N level that triggered the onset of polysaccharide accumulation was 2.3% dry weight (DW); at a cellular N concentration less than 1.4% DW, the polysaccharide synthesis stopped. Upon nitrate re-supply, mobilization of WSP occurred after 3 days. These results suggest that a two-stage cultivation process could be used to obtain large algal biomass with high water-soluble polysaccharide production: a first cultivation stage using nitrate-supplemented medium to accumulate algal biomass followed by a second cultivation stage in a nitrate-free medium for 3 to 7 days to enhance polysaccharide content in the alga.     

The regulation of cytochrome c oxidase of Rhodobacter capsulatus by light and oxygen

In order to distinguish between the regulatory effects of oxygen tension and light intensity on cytochrome c oxidase protein and enzymatic activity cells of Rhodobacter capsulatus were shifted from phototrophic (anaerobic, light) growth to aerobic-light, aerobic-dark and to anaerobic-dark conditions, respectively. During shift-experiments the formation of oxidase protein and regulation of oxidase activity was followed by immunological and enzymatic means. The results support the idea, that the formation of oxidase protein is regulated by oxygen tension and light intensity changes, whereas the regulation of oxidase activity seems only to be correlated to the oxygen tension. A DNA sequence involved in the oxygen-dependent regulation of cytochrome oxidase could be identified in the regulation-deficient oxidase mutant H41 of R. capsulatus. Immunological investigations of cytochrome c ₂ from mutant H41 demonstrated at the same time the participation of the c ₂-polypeptide in the regulation of cytochrome c oxidase.Abbreviations Bchl bacteriochlorophyll - CIE crossed immuno-electrophoresis - DMSO dimethyl sulfoxide     

Modeling of photobioreactors: Application to bubble column simulation

Photobioreactors using algae, plant cells, or photosynthetic bacteria have received considerable attention from biochemical engineers. Industry is presently engaged in developing new products and testing a new generation of algal-derived natural products such as natural dyes, polyunsaturated fatty acids and polysaccharides. The present paper is a review of some of the recent findings of the authors in the field. A mathematical representation of the growth of a photosynthetic system in an alternating light-dark regime is presented. This model integrates fluid dynamics and maintenance into the three-state "Photosynthetic Factories" model by Eilers and Peeters. The model was solved analytically and the constants were fitted to experimental data obtained in a thin film tubular reactor. The theoretical prediction that the introduction of light-dark cycles may enhance the growth was confirmed by the experimental results. The model allows predicting the collapse of cultures in photobioreactors either under light-deficit or light-excess conditions, as well as the influence of mixing on these critical phenomena. This paper presents an approach to modeling the kinetics of photosynthetic systems for photobioreactor design. Under conditions of simultaneous occurrence of photoinhibition in one region of the reactor, and photo-limitation in another, it takes into account the movement of the cells from one region to the other. The model was applied to the mathematical modeling of a 13-liter bubble column photobioreactor. Experimental data were satisfactorily fit, using the kinetic data obtained independently in the thin-film experiments. The model was extended to simulate a "farm" of photobioreactors and the results presented defining Ground Productivity, which expresses the rate of biomass production of a farm of relatively small photobioreactors per area of ground required for the installation.     

霍山石斛类原球茎二步法培养细胞生长和多糖合成的动力学研究

磷是调控类原球茎细胞生长和多糖积累的有效因素。为了获得较高的多糖产量,根据霍山石斛类原球茎生长和多糖积累的动力学特性,提出了二步培养方式,采用了补料策略,研究了其培养过程的动力学特性,并建立了相关模型。结果表明,采用二步法培养,生物量从28.7g DW/L提高到44.2g DW/L, 多糖产量从1.86g/L提高到5.22g/L,多糖含量从6.4%提高到11.9%。建立的模型基本反映了类原球茎生长和多糖积累的动力学机制。     

Competition between the green alga Scenedesmus and the cyanobacterium Synechococcus under different modes of inorganic nitrogen supply

In this study, we evaluated growth responses of the green alga Scenedesmus and the cyanobacterium Synechococcus supplied with inorganic nitrogen in different ways. A competitive situation in which nitrogen was limiting was created in mixed cultures as well as in cultures growing in the same vessel but separated by a permeable dialysis membrane. Supplying inorganic nitrogen in small pulses at a high frequency favoured the cyanobacterium Synechococcus, whereas batch additions favoured the green alga Scenedesmus. When using a large-pulse/low-frequency supply mode, the yield of the green alga was higher when ammonium was added as nitrogen source compared to when nitrate was added. By contrast, the yield of the cyanobacterium was higher in the nitrate regime. However, uptake experiments using unialgal cultures showed that both organisms depleted the medium of ammonium more rapidly than they depleted the medium of nitrate; i.e. the higher yield of the cyanobacterium in the nitrate regime than in the ammonium regime can be attributed to the effects of competition with the green alga. Since nitrate assimilation involves the consumption of reductive power, we suggest that the outcome of competition was governed by the fact that green alga was light limited and therefore better able to compete for ammonium than for nitrate. The results from the laboratory studies are discussed in relation to results from an enclosure experiment performed in Lake Erken, Sweden. In that field experiment, in which additions of both phosphate and ammonium were applied every second day to 350-l enclosures, the green algal biomass increased exponentially during an incubation period of 22 days.     

A novel method for regenerating the protoplasts of thermophilic bacilli

A range of Bacillus thermophiles was tested for the ability to be converted to protoplasts with lysozyme and subsequently, to regenerate to bacillary form. Protoplast formation was straightforward but many of the strains failed to regenerate on commonly-used media. Two medium components were found to be causing the inhibition. Growth of protoplasts as L-forms only occurred if the medium lacked phosphate. However, reversion of L-forms to bacilli was asynchronous and infrequent. Regeneration of cell walls by protoplasts/L-forms to re-establish bacillary form was greatly improved when the medium was gelled with pluronic polyol F127 in place of agar or similar polysaccharides.     

The potential of production of sulfated polysaccharides from Porphyridium

Summary The environmental conditions prevailing in Israel make marine algae an attractive crop for the production of valuable chemicals. A marine species of Porphyridium seems to fit this purpose.The unicellular red alga Porphyridium is encapsulated by a polysaccharide envelope that is present in the gel state. This polysaccharide is an acidic heteropolymer composed of sulfated sugars. It forms ionic bridges through divalent cations, thus reaching a very high molecular weight. The thickness of the polysaccharide capsule varies according to the phase of growth and the growth conditions. Its outer part dissolves in the growth medium, which becomes progressively more viscous. Sulfated polysaccharides form theramlly reversible gels similar to agar and carrageenan, which are usually extracted from marine macroalgae. These gels have been finding increasing use in commercial applications as gelling agents, thickeners, stabilizers, and emulsifiers.We have done experiments on the cultivation of a marine species of Porphyridium for the production of polysaccharides. This unicellular alga has an advantage over the macroalgae due to its relatively faster growth rate and the possibility to regulate its growth. The potential for production of the polysaccharide, both that dissolved in the external medium and that attached to the cell (including an intracellular fraction), and the effects of growth conditions on productivity were suudied in the laboratory. Porphyridium was also cultivated outdoors in seawater in 1-m² ponds and its growth potential investigated.     

Physiological responses of Aureoumbra lagunensis and Synechococcus sp. to nitrogen addition in a mesocosm experiment

Aureoumbra lagunensis is the causative organism of the Texas brown tide and is notable because it dominated the Laguna Madre ecosystem from 1990 to 1997. This species is unusual because it has the highest known critical nitrogen to phosphorus ratio (N:P) for any microalgae ranging from 115 to 260, far higher than the 16N:1P Redfield ratio. Because of its high N:P ratio, Aureoumbra should be expected to respond to N additions that would not stimulate the growth of competitors having the Redfield ratio. To evaluate this prediction, a mesocosm experiment was performed in the Laguna Madre, a South Texas coastal lagoon, in which a mixed Aureoumbra–Synechococcus (a cyanobacterium) community was enclosed in 12 mesocosms and subjected to nitrogen addition (6 controls, 6 added ammonium) for 16 days. After day 4, added nitrogen did not significantly increase Aureoumbra specific growth rate but the alga retained dominance throughout the experiment (64–75% of total cell biovolume). In control mesocosms, Aureoumbra became less abundant during the first 4 days of the experiment but rebounded by the end of the experiment and was dominant over Synechococcus. Despite the lack of a strong positive growth response, Aureoumbra did respond physiologically to N addition. By the end of the experiment, the average N:P ratio of the Aureoumbra-dominated community was 86 in the N+ treatment and 41 in the control, indicating that the alga became less N-limited in the N+ treatment. The average C:N ratio was 6.6 in the N+ treatment (8.6 in the control) and suggests that the alga was not N-limited, however, C:N ratio may not be a good indicator of nitrogen limitation since this alga can produce significant quantities of carbon-containing extracellular polysaccharides, depending on growth conditions. Both Aureoumbra cellular chlorophyll fluorescence and cell size increased in response to added N, indicating a reduction in N limitation. It appeared that the N additions were not large and/or frequent enough to stimulate Aureoumbra growth. The main competitor, the unicellular cyanobacterium Synechococcus, responded positively to the nitrogen addition by increased specific growth rate. Unlike Aureoumbra, no significant effect on Synechococcus cellular pigment fluorescence or cell size was noted. Literature data suggest that Synechococcus, like Aureoumbra, may have a critical N:P ratio much higher than 16:1, which could explain its response.     

Cultural properties of a luminous mushroom,Mycena chlorophos

Cultural conditions on mycelial growth and fruit-body formation ofMycena chlorophos were studied. The optimum temperature of the mycelial growth was 27°C and the optimum initial pH of medium was 4.0. Peptone agar medium was suitable for the spawn culture. Compost medium containing rice bran at 10% (fw/fw) was appropriate for fruitbody formation in the Petri dish. Light was essential for initiation of primordia, and low-temperature treatment induced fruit-body formation effectively. The optimum conditions for fruit-body formation were found to be the cultivation at 27°C for 4 wk and continued cultivation for 3 wk under illumination at an intensity above 0.2 lx and at 21°C after casing with moist compost powder. In the fruit-bodies obtained, the maximum photosensitive wavelength of luminescence was 522 nm and the optimum temperature for emission was 27°C. The luminescence of a fruit-body was observed for about 3 d consecutively at 21°C.     

Studies on utilization of 2-ketoglutarate,glutamate and other amino acids by the unicellular alga Cyanidium caldarium

Two strains of Cyanidium caldarium which possess different biochemical and nutritional characteristics were examined with respect to their ability to utilize amino acids or 2-ketoglutarate as substrates.One strain utilizes alanine, glutamate or aspartate as nitrogen sources, and glutamate, alanine or 2-ketoglutarate as carbon and energy sources for growth in the dark. The growth rate in the dark on 2-ketoglutarate is almost twice as high or higher than that on glutamate or alanine. During growth or incubation of this alga on amino acids, large amounts of ammonia are formed; however, ammonia formation is strongly inhibited by 2-ketoglutarate. The capacity of the alga to form ammonia from amino acids is inducible and develops fully only when the cells are grown or incubated in the presence of glutamate.By contrast, the other strain of Cyanidium caldarium cannot utilize alanine or aspartate as nitrogen sources. It utilizes glutamate only very poorly and does not excrete ammonia into the external medium. This strain is unable to utilize amino acids or 2-ketoglutarate as carbon and energy sources for heterotrophic growth.Cell-free extracts were tested for the occurrence of enzymes which could account for amino acid metabolism and ammonia formation.     

Chromatic adaptation and photoreversal in blue-green algaCalothrix clavata West

Complementary chromatic adaptation, a well-established phenomenon in some blue-green algae, has been observed inCalothrix clavata, a heterocystous blue-green alga of the family Rivulariaceae. The chromatic adaptation has been observed for fluorescent and incandescent light by measuring the absorption spectra. The material grown in fluorescent light forms more of phycoerythrin whereas more of phycocyanin tends to be formed in incandescent light. Besides this, photoreversal was observed by transferring the incandescent light grown alga to fluorescent light conditions and vice-versa. Effect of photoreversal and chromatic adaptation has also been discussed for this alga under different monochromatic light conditions. The influence of different light conditions on morphological changes, heterocysts and hormogonia formation has also been investigated. Both chromatic adaptation and photomorphogentic phenolmena in this alga show the involvement of some photoreversible (red:green) pigment.     

Observations on the morphology,reproduction and cytology of stigeoclonium pascheri (vischer) cox et bold (chaetophorales-chlorophyceae) and their bearing on the validity of the genus caespitella vischer

The present communication deals with the morphology, reproduction and cytology of Stigeoclonium pascheri (formerly described as Caespitella pascheri Vischer). Effects of various concentrations of nitrate and variations in intensity and duration of light period in 24 hr LD cycles were studied on cultures of this alga in relation to the type and extent of branching and occurrence or absence of hair formation. It is concluded that these traits are not of taxonomic significance and are influenced largely by environmental conditions. The present study shows a large overlapping of morphological characters between the genera Stigeoclonium and Caespitella and supports Cox & Bold's abandonment of the latter generic name and its merger into the genus Stigeoclonium. The chromosome number determined for this alga is n = 6 which indicates that the Indian strain of this alga is a distinct cytotype from the one originally isolated by Vischer.     

Carbohydrates of the freshwater alga Tribonema aequale. II. Preliminary photosynthetic studies with 14C

Tribonema aequale Pascher was cultured for 30 min and 2 h in Na₂ ¹⁴CO₃. An aliquot of the 2 h material was grown for a further 45 h in culture medium devoid of ¹⁴C. The alga was sequentially extracted and the respective radioactivities of the extracts were measured. Evidence is presented that not only mannitol and glucose, but also the water-soluble glucose-containing polysaccharides, are active metabolites. The polysaccharides soluble in 4% aqueous alkali appear to be continuously synthesised and that soluble in 18% alkali is apparently incorporated into the insoluble non-extractable polysaccharide during growth.     

Photobioreactor for cultivation and real‐time,in‐situ measurement of O2 and CO2 exchange rates,growth dynamics,and of chlorophyll fluorescence emission of photoautotrophic microorganisms

A detailed knowledge about the dynamics of phytoplanktonic photosynthesis and respiration is crucial for the determination of primary productivity in open oceans as well as for biotechnological applications. The dynamics are best studied in photobioreactors that are able to simulate natural conditions in such, that light can be modulated not only diurnally but also mimicking effects of solar elevation angle from sunrise to sunset, variable cloudiness, light modulation in refractory sun flecks due to water waves, or light intermittence due to turbulent flow in dense suspensions. In addition, high performance photobioreactors ought to be able to monitor in real time photosynthetic and respiratory activities as well as culture growth. Here, we demonstrate performance of a newly designed bench‐top laboratory photobioreactor that meets these needs, with a study of green alga Scenedesmus quadricauda. The algal suspension was exposed to simulated daily variations of total photosynthetic active irradiance and spectral profile, with a larger proportion of red photons in the morning and evening hours. The instrument monitored automatically the culture growth by measuring the optical densities at 735 nm and 680 nm and by measuring steady state and maximal chlorophyll fluorescence emission yields. The photochemical yields were estimated from chlorophyll fluorescence data. These widely used but rather indirect yield estimates were confronted with direct measurements of oxygen evolution and consumption quantum yields. The CO₂ fluxes in and out of the culture media as well as the dissolved CO₂ in algal suspension were also recorded. The experiments demonstrated potential of the new photobioreactor to reveal minute modulations in gas exchange rates as well as to yield data for calculation of photon requirement of oxygen evolution in the suspension volume that is key technological parameter for planning of large scale photobioreactors as well as key optimization parameter for strain selection.     

Microalgae--novel highly efficient starch producers

The freshwater alga Chlorella, a highly productive source of starch, might substitute for starch‐rich terrestrial plants in bioethanol production. The cultivation conditions necessary for maximizing starch content in Chlorella biomass, generated in outdoor scale‐up solar photobioreactors, are described. The most important factor that can affect the rate of starch synthesis, and its accumulation, is mean illumination resulting from a combination of biomass concentration and incident light intensity. While 8.5% DW of starch was attained at a mean light intensity of 215 µmol/(m² s¹), 40% of DW was synthesized at a mean light intensity 330 µmol/(m² s¹). Another important factor is the phase of the cell cycle. The content of starch was highest (45% of DW) prior to cell division, but during the course of division, its cellular level rapidly decreased to about 13% of DW in cells grown in light, or to about 4% in those kept in the dark during the division phase. To produce biomass with high starch content, it is necessary to suppress cell division events, but not to disturb synthesis of starch in the chloroplast. The addition of cycloheximide (1 mg/L), a specific inhibitor of cytoplasmic protein synthesis, and the effect of element limitation (nitrogen, sulfur, phosphorus) were tested. The majority of the experiments were carried out in laboratory‐scale photobioreactors, where culture treatments increased starch content to up to about 60% of DW in the case of cycloheximide inhibition or sulfur limitation. When the cells were limited by phosphorus or nitrogen supply, the cellular starch content increased to 55% or 38% of DW, respectively, however, after about 20 h, growth of the cultures stopped producing starch, and the content of starch again decreased. Sulfur limited and cycloheximide‐treated cells maintained a high content of starch (60% of DW) for up to 2 days. Sulfur limitation, the most appropriate treatment for scaled‐up culture of starch‐enriched biomass, was carried out in an outdoor pilot‐scale experiment. After 120 h of growth in complete mineral medium, during which time the starch content reached around 18% of DW, sulfur limitation increased the starch content to 50% of DW. Biotechnol. Bioeng. 2011; 108:766–776. © 2010 Wiley Periodicals, Inc.     

乙酸钠调控小球藻生长及代谢产物

【背景】小球藻是一种单细胞绿藻,在不同培养条件下可积累高附加值的代谢产物,这些产物可用于生产生物燃料、食品、保健品、药品等。然而这些代谢产物在藻细胞中的生产率较低且很难通过经济可行的方法将其分离,这使其工业化规模生产受到限制。【目的】研究乙酸钠对小球藻生物量的影响,并分析其对小球藻代谢产物的调控作用。【方法】通过在小球藻培养液中添加不同浓度的乙酸钠(1.0、2.0、3.0、4.0、5.0 g/L),研究其调控小球藻生长和代谢的作用机理。【结果】在添加3.0 g/L乙酸钠的培养液中,小球藻的生物量是对照组的5.2倍,尽管藻细胞中蛋白质含量无明显变化,但油脂和类胡萝卜素含量是对照组的2.4倍和1.2倍,多糖和叶绿素a含量却仅为对照组的54.6%和54.4%。【结论】乙酸钠不仅会影响藻细胞的生长,还会调控其代谢过程,这为深入探索乙酸钠在调控小球藻生长及代谢过程的作用机制提供了理论基础和技术资料。     

Photoautotrophic cultivating options of freshwater green microalgal Chlorococcum humicola for biomass and carotenoid production

Photoautotrophic cultivation of Chlorococcum humicola was performed in batch and continuous modes in different cultivating system arrangements to compare biomass and carotenoids’ concentration and their productivities. Batch result from stirred tank and airlift photobioreactors indicated the positive effect of increasing light intensity on growth and carotenoid production, whereas the finding from continuous cultivation indicated that carotenoid enhancement preferred high light intensity and nitrogen-deficient environment. The highest biomass (1.31?±?0.04?g?L^?1) and carotenoid (4.59?±?0.06?mg?L^?1) concentration as well as the highest productivities, 0.46?g?L^?1 d^?1 for biomass and 1.61?mg?L^?1 d^?1 for carotenoids, were obtained when maintaining high light intensity of 10 klx, BG-11 medium and 2% (v/v) CO₂ simultaneously, while the highest carotenoid content (4.84?mg?g^?1) was associated with high light intensity and nitrogen-deficient environment, which was induced by feed-modified BG-11 growth medium containing nitrate 20 folds lower than the original medium. Finally, the cultivating system arranged into smaller stirred tank photobioreactors in series yielded approximately 2.5 folds increase in both biomass and carotenoid productivities relative to using single airlift photobioreactor with equivalent working volume and similar operating condition.