EFFECT OF SODIUM AND NITRATE ON GROWTH OF ANABAENA FLOS-AQUAE (CYANOPHYTA)1

The growth response of a nitrogen fixing cyanobacterium, Anabaena flos-aquae (Lyng) Bréb., to sodium and nitrate was examined in batch culture under controlled laboratory conditions. Sodium (range 0-12 mg Na⁺· L^-1) enhanced growth of the cyanobacterium under nitrate-sufficient (5.7 mg NO³-N · L^-1) but not nitrate-limited (0.49 mg NO³- N · L^-1) conditions. The magnitude of the growth response was related to the nutritional history of the culture. No significant effect of sodium on nitrate utilization was observed. The increase in ambient sodium levels in many lakes may provide a competitive advantage to cyanobacteria.     

A NUMERICAL TAXONOMIC STUDY OF NOSTOC AND ANABAENA1

Thirty characteristics of 14 Nostoc and 10 Anabaena species were analyzed from previously published data. Using standard numerical taxonomic methods, simple matching coefficients were calculated and a phenogram drawn. The analysis revealed that some of the central characteristics of Nostoc are: a punctiforme stage; motile reproductive stage; plant mass with a dull to shiny luster, non-veined surface, and nonfimbriate margin; some spherical vegetative cells; no cylindrical heterocysts; and some spherical, but no cylindrical akinetes. Some of the central characteristics of Anabaena that were revealed are: no punctiforme stage; a motile vegetative stage; plant mass with a shiny luster, veined surface, and fimbriate margin; no spherical vegetative cells; some cylindrical heterocysts; and some cylindrical, but no spherical, akinetes. In general, Anabaena has larger akinetes and vegetative cells than Nostoc. Based on 30 morphological characteristics and the clustering data of the phenogram, keys were constructed for the Nostoc and Anabaena species studied. The data clearly support two separate and distinct, though similar genera and, less sharply, the separation of the 24 species. The more useful characteristics for separation of the species are size and shape of akinetes, vegetative cells, and heterocysts; color and luster of plant mass; veined plant mass surface; margin fimbriate; and shape of plant mass in nature.     

IRON STIMULATION OF PHOTOSYNTHESIS AND NITROGEN FIXATION IN ANABAENA 7120 AND TRICHODESMIUM (CYANOPHYCEAE)1

Iron availability may limit carbon and nitrogen fixation in the oceans. The freshwater cyanobacterium, Anabaena, was used as a laboratory model for the biochemical and physiological effects of iron. Increased iron nutrition, in the range of 10^?8 M to 10^?6 M resulted in increases of approximately four fold in carbon and nitrogen fixation rates. Chlorophyll concentration increased, and the relative amount of in vivo fluorescence was reduced with more iron. Natural samples of Trichodesmium, collected off Barbados and incubated with increased iron for two days, showed similar effects. Trichodesmium responded to iron additions indicating that it may be Fe limited in its natural environment. These responses to iron are consistent with the biochemical roles of iron in photosynthesis and nitrogen fixation. The results are discussed in the geochemical context of the sporadic total iron input to tropical oceans and possible implications to spatial and temporal patterns of productivity.     

固氮鱼腥藻(Anabaena azotica Ley HB 686)氢酶的存在形式和性质

Anabaena azotica HB 686氢酶有可溶性和膜结合两种存在状态,其中膜结合氢酶占总吸氢活性的67％,是吸氢酶的主要存在形式。在分离过程中,这两种氢酶在DEAE(DE—52)柱上的洗脱行为不同;此外,膜结合氨酶与氢的亲和力和吸氢能力均较强,对低温更敏感。这些差异可能与这两种氨酶的结构与生理功能不同有关。     

ISOLATION AND CHARACTERIZATION OF CYANOBACTERIA POSSESSING ANTIMICROBIAL ACTIVITY FROM THE SUNDARBANS,THE WORLD’S LARGEST TIDAL MANGROVE FOREST1

Eight obligately halophilic, euryhaline cyanobacteria from intertidal soil were isolated in artificial seawater nutrients III (ASN‐III) medium. Antimicrobial activity, 16S rRNA gene sequences, phenotypic characters as well as growth and antibiosis in response to variable salinity, temperature, phosphate concentration, and pH were studied. Minimum inhibitory concentrations (MIC) of the extracts against Staphylococcus aureus, Escherichia coli, Bacillus subtilis, Pseudomonas aeruginosa, and multiple drug‐resistant clinical isolates ranged between 0.25 and 0.5 mg · mL⁻¹. Cytotoxicity tests showed 73%–84% human colon adenocarcinoma (HT‐29/C1) cell survival at MIC values, indicating that the extracts were nontoxic. Morphologically, six cyanobacteria were assigned to the Lyngbya‐Phormidium‐Plectonema (LPP) group B, and one each was assigned to Oscillatoria and Synechocystis genera. Glycerol, mannitol, and starch supported better photoheterotrophic growth than simpler mono‐ and disaccharides. No heterocyst formation was observed when grown under nitrogen‐starved conditions. All isolates survived 7‰ salinity, grew at minimum 32‰ salinity, and showed sustained growth throughout 32‰–82‰ salinity but matured poorly in freshwater medium supplemented with 30.0 g · L⁻¹ NaCl. Antimicrobial production occurred only at 32‰ salinity. While four of the eight isolates demonstrated sustained growth at 37°C, maximum antimicrobial activity was obtained at 25°C. All strains showed maximum growth and antimicrobial elaboration at 0.04 g · L⁻¹ phosphate. All isolates thrived at pH 9.5; six grew at pH 4.5, though antimicrobial production occurred only at pH 7.5. Molecular phylogenetic analysis based on 16S rRNA gene sequences of the filamentous isolates validated the previous taxonomic affiliations established on morphological characteristics. This is the first study of antimicrobial‐producing halophilic cyanobacteria from the mangroves.     

EFFECT OF CULTURE AND BLOOM DEVELOPMENT AND OF SAMPLE STORAGE ON PARALYTIC SHELLFISH POISONS IN THE CYANOBACTERIUM ANABAENA CIRCINALIS

Paralytic shellfish poisons (PSPs) were detected in 24 of 31 bloom samples dominated by the cyanobacterium Anabaena circinalis Rabenhorst, collected from across Austraia. The ability to produce PSPs has been maintained in everal non-axenic strains of A. circinalis kept in culture, whereas strains that were non-toxin-producing when isolated have remained as such. PSPs were detected and quantified by high-performance liquid chromatography (HPLC), and the structures were confirmed by electrospray mass spectroscopy. The concentration of toxins in PSP positive samples ranged from 50 to 3400 μg.g^-1 dry weight. Toxin profiles were always dominated by the N-sulfocarbamoyl-11-hydroxysulfate C toxins, C1 and C2 (44–85 mol%), with the remainder consisting of gonyautoxins-2, −3, and −5, decarbamoylgonyautoxins-2 and −3, saxitoxin, and decarbamoylraxitoxin. N₁-_-hydroxy PSPs, commonly found in marine dinoflagellates, were absent, suggesting that A. circinalis lacks the enzyme responsible for N₁-_-hydroxylation. On a dry weight basis, the amount of toxin in cultured Anabaena circinalis (strain ACMB06)rose significantly (P < 0.05)over time from 570 to 3400 μg.g.^-1 cells in late stationary phase. However, there was no significant trend in cellular toxin quota (toxin per cell) over the life of the culture; this may be explained by variation in cell mass. On average, batch cultures of Anabaena circinalis contained 19% extracellular toxin, which increased slightly over the growth cycle and had a composition similar to that of the intracellular toxins. As cultures aged, the formation of decarbamoyl toxins and increases in theα-/β-epimer ratios of C toxins and gonyautoxins were observed. The variation in these components during stationary phase in culture was sufficient to explain the variation in relative PSP composition observed among natural bloom samples. Because decarbamoylgonyautoxins are much more toxic than C toxins on a molar basis, these transformations also lead to an increase in toxicity of the sample or bloom over time. The transformations of PSPs, which occur during aging and sample storage, render the comparison of PSPs by HPLC unreliable for phenotyping Anabaena circinalis, unless strains are cultured, harvested, and analyzed under standard conditions.     

SIDEROCHROMES IN CYANOPHYCEAE: ISOLATION AND CHARACTERIZATION OF SCHIZOKINEN FROM ANABAENA SP.1

Schizokinen, a high affinity iron transport compound (siderochrome), previously described from Bacillus megaterium, has been isolated and characterized from low-iron cultures of a bluegreen alga, Anabaena sp. No conclusive evidence for production of an iron-repressible hydroxamate or catechol type siderochrome was observed in certain other bluegreen algae, such as Anabaena cylindrica Lemm., Coccochloris peniocystis (Kütz.) Drouet & Daily, Anacystis sp., Gloeocapsa alpicola (Lyng.) Born. or Chroococcus sp.     

PROTEIN TURNOVER AND HETEROCYST DIFFERENTIATION IN THE CYANOBACTERIUM ANABAENA VARIABILIS1

Under conditions of starvation for fixed nitrogen, cells of the filamentous cyanobacterium Anabaena variabilis Kütz, degrade much of their protein prior to heterocyst differentiation. Cells starved for a source of fixed nitrogen initially degraded about 2% of their protein per hour; by 24 h after nitrogen stepdown about 40% of the protein was degraded. Most of the acid-soluble radiolabeled material was excreted into the medium. Proteolysis was completely inhibited by chloramphenicol, by cyanide, or in the dark, hut was only partially inhibited in the presence of dichlorophenyl dimethylurea. Methionine sulfoximine (MSX) (an inhibitor of glutamine synthetase) in the presence of ammonia caused heterocysts to form. MSX treated cells degraded protein; however, the amount of protein degraded was much less than in cells starved for ammonia. Glutamine, which can serve as a nitrogen source for this strain, did not prevent starvation-induced proteolysis and did not prevent the differentiation of heterocysts.     

STORAGE OF PHOSPHORUS IN NITROGEN-FIXING ANABAENA FLOS-AQUAE (CYANOPHYCEAE)1

P accumulation and metabolic pathway in N₂-fixing Anabaena flos-aquae (Lyngb.) Bréb were investigated in P-sufficient (20 μMP) and P-limited (2 μMP) turbidostats in combined N-free medium. The cyanobacterium grew at its maximum rate (μ_max, 1.13 d^?1) at the high P concentration and at 65% of μ_max under P limitation, with total cell P concentrations (Q_P) at steady states of 12.0 and 5.2 fmol·cell^?1, respectively. At steady state, polyphosphates (PP_i) accounted for only 3% of Q_P (0.4 fmol·cell^?1) in P-rich cells. Its concentration in P-limited cells was 5.8% (0.3 fmol·cell^?1). On the other hand, sugar P was very high at 22% of Q_P in P-rich cells and was undetectable in P-limited cells. Pulse chase experiments with ³²P showed that P-rich cells initially incorporated the labeled P into the acid-soluble PP_i fraction within the first few minutes and to a lesser extent into nucleotide P. Radioactivity in the PP_i then declined rapidly with concomitant increases in sugar P and nucleotide P fractions. In contrast, in P-limited cells, no radiolabel was detected in acid-soluble PP_i, and ³²P was initially incorporated into nucleotide P, sugar P, and ortho P fractions. The latter two fractions then subsequently declined. Therefore, under N₂-fixing conditions the cyanobacteria appeared to store P as sugar P and also utilize P through different pathways under P-rich and -limited conditions. When nitrate was supplied as the N source under P-sufficient conditions, PP_i accounted for about 15% of steady-state Q_P, but no sugar P was detected. Therefore, the same organism stored P in different cell P fractions depending on its N sources.     

POLYPHASIC CHARACTERIZATION OF THREE STRAINS OF ANABAENA RENIFORMIS AND APHANIZOMENON APHANIZOMENOIDES (CYANOBACTERIA) AND THEIR RECLASSIFICATION TO SPHAEROSPERMUM GEN. NOV. (INCL. ANABAENA KISSELEVIANA)1

Occurrences of rare cyanobacteria Anabaena reniformis Lemmerm. and Aphanizomenon aphanizomenoides (Forti) Horecká et Komárek were recently detected at several localities in the Czech Republic. Two monoclonal strains of An. reniformis and one strain of Aph. aphanizomenoides were isolated from distant localities and different sampling years. They were characterized by a combination of morphological, genetic, and biochemical approaches. For the first time, partial 16S rRNA gene sequences were obtained for these morphospecies. Based on this gene, all of these strains clustered separately from other planktonic Anabaena and Aphanizomenon strains. They appeared in a cluster with Cylindrospermopsis Seenaya et Subba Raju and Raphidiopsis F. E. Fritsch et M. F. Rich, clustered closely together with two An. kisseleviana Elenkin strains available from GenBank. A new generic entity was defined (Sphaerospermum gen. nov., with the type species S. reniforme, based on the traditional species An. reniformis). These results contribute significantly to the knowledge base about genetic heterogeneity among planktonic Anabaena–like and Aphanizomenon–like morphospecies. Accordingly, the subgenus Dolichospermum, previously proposed for the group of planktonic Anabaena, should be revaluated. Secondary metabolite profiles of the An. reniformis and Aph. aphanizomenoides strains differed considerably from 17 other planktonic Anabaena strains of eight morphospecies isolated from Czech water bodies. Production of puwainaphycin A was found in both of the An. reniformis strains. Despite the relatively short phylogenetic distance from Cylidrospermopsis, the production of cylindrospermopsin was not detected in any of our strains.     

BIOLOGICAL AVAILABILITY OF IRON TO THE FRESHWATER CYANOBACTERIUM ANABAENA FLOS‐AQUAE1

Iron acquisition from various ferric chelates and colloids was studied using iron‐limited cells of Anabaena flos‐aquae (Lyng.) Brèb UTEX 1444, a cyanobacterial strain that produces high levels of siderophores under iron limitation. Various chelators of greatly varying affinity for Fe³⁺ (HEDTA, EDDHA, desferrioxamine mesylate, HBED, 8‐hydroxyquinoline) were assayed for the degree of iron acquisition by iron‐limited cyanobacterial cells. Iron uptake rates (measured by graphite furnace atomic absorption spectrometry) varied approximately inversely with calculated [Fe³⁺] (calculated as pFe) and decreased with increasing chelator‐to‐iron ratio. No iron uptake was observed when Fe³⁺ was chelated with HBED, the strongest of the tested chelators. Iron‐limited Anabaena cells were able to take up iron from 8‐hydroxyquinoline (oxine or 8HQ), a compound sometimes used to quantify aquatic iron bioavailability. Iron bound to purified humic acid was poorly available but did support some growth at high humic acid concentrations. These results suggest that for cyanobacteria, even tightly bound iron is biologically available, including to a limited extent iron bound to humic acids. However, iron bound to some extremely strong chelators (e.g. HBED) is likely to be biologically unavailable.     

三甲基月桂基硫酸钠（TLS）在蓝细菌核酸提取中的作用

本文首次把三甲基月桂基硫酸钠(TLS)应用于蓝细菌(蓝藻)总核酸的提取。在用溶菌酶破坏满江红鱼腥藻的细胞壁后,再用TLS分别取代常用法中的各种试剂,即:Triton X-100 Sarkosyl,蛋白酶K,总核酸产率分别是常用法的98.9％,47.3％,103.8％。用TLS取代蛋白酶K处理不同生长期的满江红鱼腥藻,总核酸产率随生长期延长而降低。TLS取代蛋白酶K提取的DNA和常用法提取的DNA,都能被限制性内切酶EcoR I水解。用光学显微镜观察到,满江红鱼腥藻经TLS处理后,丝状体断裂,细胞破裂成碎片。在扫描电子显徽镜下,未经TLS处理的细胞表面光滑平整,而经TLS处理的细胞表面有鳞片状突起、皱褶、凹陷,甚至穿孔。     

POLYSACCHARIDES FROM THE ENVELOPES OF HETEROCYSTS AND SPORES OF THE BLUE-GREEN ALGAE ANABAENA VARIABILIS AND CYLINDROSPERMUM LICHENIFORME1

The polysaccharides from the envelopes of heterocysts of Cylindrospermum licheniforme Kütz., and of heterocysts and spores of Anabaena variabilis Kütz., like those from the differentiated cells of Anabaena cylindrica Lemm., have a 1,3-linked backbone consisting of glucosyl and mannosyl residues in a molar ratio of approximately 3:1. As is the case with A. cylindrica the polysaccharides from A. variabilis and from the heterocysts of C. licheniforme have terminal xylosyl and galactosyl residues as side branches. In addition, the polysaccharide from C. licheniforme resembles that from A. cylindrica in having terminal mannosyl residues as side branches (absent from A. variabilis). The polysaccharides from A. variabilis resemble that from A. cylindrica in having glucose-containing side branches (absent from the heterocyst polysaccharide from C. licheniforme), but in contrast to the polysaccharides from the other two species they also have terminal arabinosyl residues as side branches. All of the polysaccharides mentioned appear to be structurally related; we present tentative structures for those not previously investigated. In contrast, the envelope of spores of C. licheniforme contains only a largely 4-linked galactan. The bulk of this envelope is not polysaccharide in nature, and contains aromatic groups.     

AKINETE DIFFERENTIATION IN ANABAENA CIRCINALIS (CYANOPHYTA)1

Three lines of evidence established conclusively that phosphorus limitation triggered akinetes to differentiate in Anabaena circinalis Rabenhorst. First, akinetes differentiated when phosphorus was limited, but not when nitrogen, inorganic carbon, iron, trace elements, or light were limited, or when dissolved oxygen concentration was increased. In the phosphorus limitation experiment, akinetes appeared first in the 0 mg P-L^?1 cultures, and the higher the initial concentration of phosphorus was, the longer it took for akinetes to differentiate. Second, akinete differentiation commenced when Q_p fell to the same critical concentration in all cultures. The critical Q_p for akinete differentiation in A. circinalis was 0.3-0.45 pg P·cell^?1, and there was no significant difference between cultures grown with 0.6, 0.2, 0.06, or 0 mg P · L^?1 (F= 5.48, of = 3, P > 0.05). Similarly, there were no significant differences between P cultures in internal cellular soluble reactive phosphorus (SRP) concentration (F= 0.63, df = 3, P > 0.05) or external SRP per cell in the medium (F= 5.16, df= 3, P > 0.05) when akinete differentiation commenced. Both were between 0.01 and 0.07 pg SRP-cell^?1. A thorough literature search indicates that this information has not been reported previously. The third line of evidence came from electron micrographs, which illustrated that polyphosphate was present in trichomes prior to akinete differentiation but was absent in trichomes with akinetes indicating that phosphorus reserves were depleted when akinetes differentiated. Lipid globules (carbon reserve) and cyanophycin granules (nitrogen reserve) increased in number in trichomes with akinetes, compared to trichomes without akinetes. Thus, the ratio of internal P:C:N was different in trichomes with akinetes compared to trichomes without akinetes and may be important in activating akinete-differentiating genes.     

SIDEROPHORE‐INDEPENDENT IRON UPTAKE BY IRON‐LIMITED CELLS OF THE CYANOBACTERIUM ANABAENA FLOS‐AQUAE1

Iron acquisition by iron‐limited cyanobacteria is typically considered to be mediated mainly by siderophores, iron‐chelating molecules released by iron‐limited cyanobacteria into the environment. In this set of experiments, iron uptake by iron‐limited cells of the cyanobacterium Anabaena flos‐aquae (L.) Bory was investigated in cells resuspended in siderophore‐free medium. Removal of siderophores decreased iron‐uptake rates by ～60% compared to siderophore‐replete conditions; however, substantial rates of iron uptake remained. In the absence of siderophores, Fe(III) uptake was much more rapid from a weaker synthetic chelator [N‐(2‐hydroxyethyl)ethylenediamine‐N,N′,N′‐triacetic acid (HEDTA); log K_cond = 28.64 for Fe(III)HEDTA(OH)^?] than from a very strong chelator [N,N′‐bis(2‐hydroxybenzyl)‐ethylenediamine‐N,N′‐diacetic acid (HBED); log K_cond = 31.40 for Fe(III)HBED^?], and increasing chelator:Fe(III) ratios decreased the Fe(III)‐uptake rate; these results were evident in both short‐term (4 h; absence of siderophores) and long‐term (116 h; presence of siderophores) experiments. However, free (nonchelated) Fe(III) provided the most rapid iron uptake in siderophore‐free conditions. The results of the short‐term experiments are consistent with an Fe(III)‐binding/uptake mechanism associated with the cyanobacterial outer membrane that operates independently of extracellular siderophores. Iron uptake was inhibited by temperature‐shock treatments of the cells and by metabolically compromising the cells with diphenyleneiodonium; this finding indicates that the process is dependent on active metabolism to operate and is not simply a passive Fe(III)‐binding mechanism. Overall, these results point to an important, siderophore‐independent iron‐acquisition mechanism by iron‐limited cyanobacterial cells.     

RESPONSE OF ANABAENA FLOS-AQUAE (CYANOPHYTA) NITROGENASE ACTIVITY TO SUDDEN PHOSPHATE DEPRIVATION1

Cultures of Anabaena flos-aquae (Lyng.) Breb. Were used to determine changes in nitrogenase activity (acetylene reduction) after external concentrations of phosphorus were lowered. Two days following immersion in phosphorus-free medium, nitrogenase activity (NA) had doubled and required 8 days to return to time zero levels. Subsequent long-term experiments showed that concentrations of soluble reactive phosphorus (SRP) released from the algae transferred into the –P medium reached maximum levels by day 3 and returned to initial low values by days 7–10. NA was always highest during this SRP release-reassimilation phase but steadily decreased after reassimilation was complete. Day 56 NA was 5–14% of initial activity. The data support the hypothesis that heterocyst and vegetative cell ATP pools are discrete and suggest that the short-term effects of phosphorus removal as an aquatic restoration technique need further study.     

UNRAVELING MOLECULAR DIVERSITY AND PHYLOGENY OF APHANIZOMENON (NOSTOCALES,CYANOBACTERIA) STRAINS ISOLATED FROM CHINA1

Fifty‐three strains of the genus Aphanizomenon isolated from Chinese waters were employed to conduct morphological examination and sequencing of the 16S rRNA gene, rbcLX (RUBISCO), and cpcBA‐IGS gene regions. Based on morphological characteristics, the examined strains were divided into three morphotypes [Aph. flos‐aquae Bréb. ex Bornet et Flahault, Aph. gracile Lemmerm., and Aph. issatchenkoi (Usacer) Proshk.‐Lavr.]. Phylogenetic analysis based on 16S rRNA and rbcLX showed that Aphanizomenon strains could be divided into three main clades (Clade A of Aph. flos‐aquae, Clade B of Aph. gracile, and Clade C of Aph. issatchenkoi), but two additional clades formed by Aph. ovalisporum and Aph. aphanizomenoides were detected in the 16S rDNA‐based topology. All Aph. issatchenkoi strains contained an additional 175 nucleotides from the 779 to 954 nucleotide location in rbcLX region, compared with strains of Aph. flos‐aquae and Aph. gracile. The cpcBA‐IGS‐based phylogenetic tree revealed that Aph. issatchenkoi strains were not discriminated from Aph. flos‐aquae strains; however, a concatenated alignment of 16S rDNA, rbcLX, and cpcBA‐IGS led to the three distinct clades (Aph. flos‐aquae, Aph. gracile, and Aph. issatchenkoi, respectively). It is suggested that the taxonomic revision of Aphanizomenon and Anabaena genera is required to be performed by employing multilocus sequence analysis and polyphasic studies.     

鱼腥藻7120异形胞分化的调节及细胞蛋白的变化

分离了有固氮活性的异形胞,它的可溶部分和膜部分的吸收光谱与营养细胞明显不同。SDS凝胶电泳图谱表明,营养细胞中存在的可溶蛋白,在异形胞中有一半左右被降解,最明显的是藻蓝蛋白。异形孢具有与营养细胞共同的肽带,但也合成了一些新的多肽。异形胞可溶蛋白有五条最主要的肽带,表观分子量约为73K,54K,48K,4lK和34K。膜蛋白中至少有2个多肽带(4lK,35K)在营养细胞膜蛋白中是缺少的。     

TRANSPARENT EXOPOLYMER PARTICLES FORMATION FROM CAPSULES OF ANABAENA SPIROIDES (CYANOBACTERIA) IN CULTURE1

We report the production of large numbers of transparent exopolymer particles (TEP) from polysaccharidic capsules of Anabaena spiroides Kleb. in cultures. Two biotic pathways of TEP formation were observed: (1) fragmentation of small portions of the capsules, which occurred throughout the growth phases; and (2) transformation of the whole polysaccharidic capsules into TEP, following cellular lyses in the aging culture. Photographic documentation of these processes was performed after staining small aliquots of the samples with Alcian Blue and negative staining with India ink. Concentrations of TEP were determined in distinct culture growth phases using semiquantitative Alcian Blue staining. Concentrations of TEP increased throughout the experimental time, while Alcian Blue remaining in solution decreased. Decreasing concentrations of chl a indicated cellular death, and by the end of the experiment, TEP formed by both pathways accumulate in the culture medium. These results show that virtually all dead chains of A. spiroides are transformed into TEP in the aged culture.