BIODEGRADATION OF PHTHALATE ESTERS BY CYANOBACTERIA1

Phthalate esters (PEs) are endocrine‐disrupting pollutants that are ubiquitous in the environment and can be degraded by microorganisms. In this study, we investigated the kinetics and pathway of biodegradation of di‐n‐butyl phthalate (DBP), diethyl phthalate (DEP), and dimethyl phthalate (DMP) by cyanobacteria Anabaena flos‐aquae G. S. West (strain 4054) and two strains of Microcystis aeruginosa (Kütz.) Kütz. (strain 2396 and strain SM). Gas chromatography/mass spectroscopy (GC/MS) and a deuterium‐labeled compound were used to analyze the degrading intermediates. The findings revealed that all three organisms were capable of metabolizing PE, and that among these organisms, A. flos‐aquae achieved the highest degradation. Additionally, the biodegradation of DBP, DEP, and DMP followed first‐order kinetics. Moreover, the results of the enzymatic study suggested that PE was degraded through transesterification on the side chains rather than deesterification. Finally, experiments using deuterium‐labeled DBP showed that there were two degradation pathways: C₁₆→ C₁₄→ C₁₂→ C₁₀→ C₈ and C₁₆→ C₁₅→ C₁₃→ C₁₁→ C₉. Based on our results, the biodegradation pathway of PE for cyanobacteria was suggested.     

水华蓝藻产毒特性的PCR检测法

特异引物对（TOX 1P／1F;TOX 2P／2F）用于检测微囊灌毒素合成酶基因mcyB片段在38种水华蓝藻中的分布情况。结果显示,所有能产生微囊灌毒素的微囊藻都有特异扩增条带,非产毒株则没有,几种常规的毒性检测方法验证了PCR方法所获结果的准确性。本研究发展了以全细胞PCR法检测mcyB片断,说明全细胞PCR检测法适用于不同来源的蓝藻材料。结果证明以DNA为基因鉴别产毒和非产毒微囊藻及其他水华蓝藻的方法是可行的和实用的。     

INFLUENCE OF IRON LIMITATION AND NITROGEN SOURCE ON GROWTH AND SIDEROPHORE PRODUCTION BY CYANOBACTERIA1

To characterize the mobilization and uptake of iron by cyanobacteria, 14 species were screened for ability to scavenge iron in a competitive system. The cyanobacteria exhibited a range of growth responses to iron limitation which could be separated into three groups, and a representative species from each group was chosen for further study. Effects of iron-limitation on growth and siderophore production of Anacystis nidulans R2, Anabaena variabilis ATCC 29413, and Plectonema boryanum UTEX 581 were determined. Both A. nidulans R2 and A. variabilis showed a reduced rate of growth with decreased available iron concentration (PFe 17–19). Growth rates increased with further reduction in the level of available iron (pFe 20 to pFe 21). The increase in growth rate occurred at the same available iron concentration as the initiation of extracellular siderophore production. In contrast, the growth of P. boryanum decreased with decreasing available iron levels. No siderophore production was detected from P. boryanum cultures. The growth kinetics of siderophore-producing species differ from traditional nutrient-limited growth kinetics and clearly reflect the presence of a high affinity, siderophore-mediated iron transport system in A. nidulans R2 and A. variabilis. Iron-limited growth kinetics more similar to traditional nutrient-limited growth kinetics were found in P. boryanum. The available nitrogen source influenced amount of siderophore produced and concentration of available iron which induced siderophore production. Siderophores were produced at high iron concentrations (pFe 18) when A. variablilis cultures were grown in the absence of combined nitrogen source. When nitrate was supplied to the culture, iron concentrations had to be reduced to pFe 20 before siderophores were produced. Cells grown on nitrogen also produced greater than two times the amount of siderophore compared with nitrate grown cells. This may be indicative of an increased demand for iron by nitrogen fixing A. variabilis Cultures.     

POLYSACCHARIDE RELEASE BY APHANOTHECE HALOPHYTICA INHIBITS CYANOBACTERIA/CLAY FLOCCULATION1

Many microalgae release polysaccharides, but the effects of the polysaccharides on mutual flocculation of microalgae and clay in aquatic environments have not been well studied. Aphanothece halophytica Frémy is a bloom‐forming cyanobacterium in salterns and can release large amounts of polysaccharide (AH‐RPS). In the present study, we investigated the effect of AH‐RPS on mutual flocculation of cyanobacterium and clay and further explored the mechanisms by which AH‐RPS affected mutual flocculation. We determined that AH‐RPS possessed clay‐dispersing activity as defined as the ability to inhibit the flocculation and sedimentation of clay suspensions in water. Supplementation of AH‐RPS in cyanobacterial cell suspensions and in the culture media containing the same kaolin clay concentration dose dependently decreased flocculation of cyanobacterial cells and increased clay‐dispersing activity. These findings indicate that the clay‐dispersing activity of AH‐RPS was related to its inhibitory effect on mutual flocculation of cyanobacterial cells and clay particles. Moreover, the clay‐dispersing activity of AH‐RPS was stable from pH 3 to pH 10 and was increased by adding NaCl, MgCl₂, CaCl₂, or low concentrations of KCl (up to 0.4 M). Taken together, our data suggest that AH‐RPS might maintain its clay‐dispersing activity and inhibit mutual flocculation of microalgae and suspended clay in saltern brine.     

NOTE ESTABLISHMENT OF AXENIC CULTURES OF ANABAENA FLOS-AQUAE AND APHANOTHECE NIDULANS (CYANOBACTERIA) BY LYSOZYME TREATMENT

A new method using lysozyme for the production of axenic cultures of Anabaena flos-aquae De Brebisson and Aphanothece nidulans P. Richter was developed. Cyanobacterial growth was not inhibited at concentrations up to 1.2 g·L⁻¹ of lysozyme, whereas the growth of heterotrophic bacteria was suppressed. At concentrations up to 0.8 g·L⁻¹ of lysozyme, ampicillin caused a reduction of heterotrophic bacteria. The axenic cultures of these strains were acquired through a simple treatment using 1.0 g·L⁻¹ of lysozyme without ampicillin. These cyanobacteria resisted digestion by lysozyme at our experimental concentrations, whereas bacteria were digested selectively. This method of purification seems to be especially useful with cyanobacterial species that are sensitive to antibiotics or other germicidal agents.     

LIGHT ACTION SPECTRA OF N2 FIXATION BY HETEROCYSTOUS CYANOBACTERIA FROM THE BALTIC SEA1

An on‐line, laser photo‐acoustic, trace gas detection system in combination with a stepper motor‐controlled monochromator was used to record semicontinuous light action spectra of nitrogenase activity in heterocystous cyanobacteria. Action spectra were made of cultures of Nodularia spumigena Mertens ex Bornet & Flahault, Aphanizomenon flos‐aquae Ralfs ex Bornet & Flahault, and Anabaena sp. and from field samples of a cyanobacterial bloom in the Baltic Sea. Nitrogenase activity was stimulated by monochromatic light coinciding the red and blue peaks of chl a, the phycobiliproteins phycocyanin (allophycocyanin) and phycoerythrin, and several carotenoids. Because nitrogenase is confined to the heterocyst, it was concluded that all photopigments must have been present in these cells, were involved in light harvesting and photosynthesis, and supplied the energy for N₂ fixation. The species investigated showed marked differences in their nitrogenase action spectra, which might be related to their specific niches and to their success in cyanobacterial blooms. Moreover, light action spectra of nitrogenase activity shifted during the day, probably as the result of changes in the phycobiliprotein content of the heterocyst relative to chl a. Action spectra of nitrogenase and changes in pigment composition are essential for the understanding of the competitive abilities of species and for the estimation of N₂ fixation by a bloom of heterocystous cyanobacteria.     

INDUCTION OF VACUOLATED SPHEROPLASTS AND ISOLATION OF VACUOLES IN CYANOBACTERIA1

There is still a great deal of debate about whether cyanobacteria contain vacuoles. This might in part reflect our limited ability to isolate vacuoles. We found and isolated vacuoles from different cyanobacteria during spheroplast preparation. Lysozyme treatment induced two kinds of spheroplasts: vacuolated spheroplasts and nonvacuolated spheroplasts. Upon breakage in distilled water, vacuolated spheroplasts released transparent, spherical, and colorless vacuoles with diameters ranging from 2.3 to 16 μm. Large vacuoles could be generated by fusion of two or three small vacuoles. Additionally, large vacuoles also could engulf small ones or other cellular bodies. The isolated vacuoles could tolerate hypotonic condition, and some could be drawn into a thread. Nonvacuolated spheroplasts released few vacuoles after breaking apart. This successful confirmation and isolation of vacuoles will allow studies of the origin and function of cyanobacterial vacuoles.     

PRODUCTION OF SECONDARY METABOLITES BY FILAMENTOUS TROPICAL MARINE CYANOBACTERIA: ECOLOGICAL FUNCTIONS OF THE COMPOUNDS

Cyanobacterial blooms are becoming more common in many reef habitats. The broadly acting feeding deterrent compound ypaoamide, produced by a mixed cyanobacterial assemblage, has been linked to bloom formation and mass fish die-offs ( Siganus argenteus and Siganus spinus ) in Guam. Specific metabolites produced by Lyngbya majuscula Gomont act as both feeding attractants to the specialist herbivore Stylocheilus longicauda , and as effective feeding deterrents to generalist fishes. Two-dimensional TLC (2D-TLC) analysis of cyanobacterial crude extracts was used to select chemically distinct populations (chemotypes) of bloom-forming filamentous cyanobacteria for chemical and ecological evaluation. Crude extracts produced by different species, chemotypes, and chemically distinct Micronesian marine cyanobacterial assemblages deter feeding activity of generalist reef herbivores. The ecological function of cyanobacterial secondary metabolites, especially as related to diversity of compound production and the relationship of metabolite production to bloom formation is discussed.     

RELEASE OF CARBOHYDRATES AND PROTEINS BY A SUBTROPICAL STRAIN OF RAPHIDIOPSIS BROOKII (CYANOBACTERIA) ABLE TO PRODUCE SAXITOXIN AT THREE NITRATE CONCENTRATIONS1

Raphidiopsis brookii P. J. Hill (cyanobacteria) was isolated from a small subtropical eutrophic pond (Biguá Pond) located in the grounds of Rio Grande University in the extreme south of Brazil, following a toxic bloom of this species. Growth, saxitoxin production, and release of carbohydrates and protein were monitored at three sodium nitrate concentrations (500, 1,000, and 1,500 μM), from inoculation up to the stationary growth phase. Growth was monitored by determining the biovolume, chl content, and trichome count. Growth was better described in terms of biovolume and chl measurements, because trichome fragmentation was observed to increase at the stationary growth phase. Carbohydrates and proteins were released in small amounts during most of the experiment, with a significant increase during the stationary phase. Extracellular polysaccharides were essentially composed of glucose, galactose, N‐acetyl‐glucosamine, mannose, xylose, rhamnose, arabinose, and fucose. The relative proportions of these units showed no significant variation during growth. Small quantities of extracellular free carbohydrates were also detected, and only fucose was released in significant amounts at the lowest nitrate concentration (500 μM). R. brookii produced both saxitoxin and dc‐saxitoxin, the former at four times the rate of the latter. This was the first study demonstrating saxitoxin production and the release of both carbohydrate and protein by R. brookii.     

STRESS‐INDUCED FILAMENT FRAGMENTATION OF CALOTHRIX ELENKINII (CYANOBACTERIA) IS FACILITATED BY DEATH OF HIGH‐FLUORESCENCE CELLS1

Irradiance power and spectral composition as well as nutrient availability strongly influence differentiation of filamentous cyanobacteria. When monitoring the life cycle of Calothrix elenkinii Kossinsk., we found that low nitrogen concentration and growth under green light led to a transient appearance of high‐fluorescence cells that rapidly bleach and disintegrate, thus breaking the parental filament into shorter parts. The dynamics of the process were monitored in a microscope growth chamber by measuring transmission and chl fluorescence of individual cells by a high‐sensitivity camera. Typically, the high‐fluorescence cells appeared near the center of the parental trichome signaled by a rapid 2‐ to 3‐fold rise in their fluorescence emission. By measuring the fluorescence excitation spectra with resolution of individual cells, we showed that the elevated fluorescence emission was largely due to a high absorption by phycoerythrin and energy transfer to chl. Typically, after no more than 20 min, the fluorescence abruptly disappeared with transmission images, indicating loss of pigmentation. The bleaching was a natural process that was not caused by the measuring light. Depending on the mechanical strain, the cell bleaching was followed by breaking of the parental filament. We propose that the high‐fluorescence cells appear as a phase of programmed cell death, allowing the fragmented filaments to escape from unfavorable environmental conditions.     

RELATIONSHIP BETWEEN THE NUMBER OF DIVIDING AND NONDIVIDING CELLS OF CYANOBACTERIA IN NORTH ATLANTIC PICOPLANKTON1

In the North Atlantic over a wide geographic region that includes various oceanic regimes and a temperature range from 10 to 22° C, an increase in the number of nondividing Synechococcus cells (X) was generally accompanied by a greater-than-proportional increase in the number of dividing cells (Y). As a result, the fraction of dividing cells (FDC = Y · (Y + X)^?1) was positively related to population size (Y + X). Recognizing that FDC is generally greater in a rapidly growing population than in a slowly growing one, our empirical finding implies a positive correlation between specific growth rate and standing stock for Synechococcus. One notable exception occurred during winter (T < 5°C) in a eutrophic coastal embayment when a decrease in cell abundance was not matched by a decrease in FDC.     

A SIMPLE METHOD FOR THE ISOLATION AND ENUMERATION OF SPARSE POPULATIONS OF CYANOBACTERIA IN ESTUARINE WATERS1

A simple, sensitive assay method for the isolation and enumeration of sparse populations of cyanobacteria in an estuarine system is described. The method, based on the standard membrane-filter plate count technique, differentiates between viable and nonviable cells. It was found that an estuarine water-based agar medium was the most suitable medium for isolation of cyanobacteria. Because of the restricted nature of colony development, isolation of individual species is easily accomplished.     

EFFECT OF TEMPERATURE ON THE SENSITIVITY OF NITROGENASE TO OXYGEN IN TWO HETEROCYSTOUS CYANOBACTERIA1

The effect of temperature and oxygen on nitrogenase activity in two heterocystous cyanobacteria, Anabaena variabilis Kütz. ATCC29413 and Nostoc sp. PCC7120, was investigated. The cyanobacteria were grown under a 12:12 light:dark (L:D) cycle at 27°C and were subsequently exposed to different temperatures (27, 36, 39, and 42°C) at different steady‐state O₂ concentrations (20, 10, 5, 0%). Light response curves of nitrogenase activity were recorded under each of these conditions using an online acetylene reduction assay combined with a sensitive laser photoacoustic ethylene detection method. The light response curves were fitted with the rectangular hyperbola model from which the model parameters N_m, N_d, and α were derived. In both strains, nitrogenase activity (N_tot = N_m + N_d) was the highest at 39°C–42°C and at 0% O₂. The ratio N_tot/N_d was 4.1 and 3.1 for Anabaena and Nostoc, respectively, indicating that respectively 25% and 33% of nitrogenase activity was supported by respiration (N_d). N_tot/N_d increased with decreasing O₂ concentration and with increasing temperature. Hence, each of these factors caused a relative increase in the light‐driven nitrogenase activity (N_m). These results demonstrate that photosynthesis and respiration both contribute to nitrogenase activity in Anabaena and Nostoc and that their individual contributions depend on both O₂ concentration and temperature as the latter may dynamically alter the flux of O₂ into the heterocyst.     

GEITLERINEMA SPECIES (OSCILLATORIALES,CYANOBACTERIA) REVEALED BY CELLULAR MORPHOLOGY,ULTRASTRUCTURE, AND DNA SEQUENCING1

Geitlerinema amphibium (C. Agardh ex Gomont) Anagn. and G. unigranulatum (Rama N. Singh) Komárek et M. T. P. Azevedo are morphologically close species with characteristics frequently overlapping. Ten strains of Geitlerinema (six of G. amphibium and four of G. unigranulatum) were analyzed by DNA sequencing and transmission electronic and optical microscopy. Among the investigated strains, the two species were not separated with respect to cellular dimensions, and cellular width was the most varying characteristic. The number and localization of granules, as well as other ultrastructural characteristics, did not provide a means to discriminate between the two species. The two species were not separated either by geography or environment. These results were further corroborated by the analysis of the cpcB‐cpcA intergenic spacer (PC‐IGS) sequences. Given the fact that morphology is very uniform, plus the coexistence of these populations in the same habitat, it would be nearly impossible to distinguish between them in nature. On the other hand, two of the analyzed strains were distinct from all others based on the PC‐IGS sequences, in spite of their morphological similarity. PC‐IGS sequences indicate that these two strains could be a different species of Geitlerinema. Using morphology, cell ultrastructure, and PC‐IGS sequences, it is not possible to distinguish G. amphibium and G. unigranulatum. Therefore, they should be treated as one species, G. unigranulatum as a synonym of G. amphibium.     

SHIFT FROM CHLOROPHYTES TO CYANOBACTERIA IN BENTHIC MACROALGAE ALONG A GRADIENT OF NITRATE DEPLETION1

A survey of the spatial distribution of benthic macroalgae in a fluvial lake of the St. Lawrence River (Lake Saint‐Pierre, Quebec, Canada) revealed a shift in composition from chlorophytes to cyanobacteria along the flow path of nutrient‐rich waters originating from tributaries draining farmlands. The link between this shift and changes in water quality characteristics was investigated by sampling at 10 sites along a 15 km transect. Conductivity, current, light extinction, total phosphorus (TP; >25 μg P · L^?1), and ammonium (8–21 μg N · L^?1) remained fairly constant along the transect in contrast to nitrate concentrations, which fell sharply. Filamentous and colonial chlorophytes [Cladophora sp. and Hydrodictyon reticulatum (L.) Bory] dominated in the first 5 km where nitrate concentrations were >240 μg N · L^?1. A mixed assemblage of chlorophytes and cyanobacteria characterized a 1 km transition zone where nitrate decreased to 40–80 μg N · L^?1. In the last section of the transect, nitrate concentrations dropped below 10 μg N · L^?1, and cyanobacteria (benthic filamentous mats of Lyngbya wollei Farl. ex Gomont and epiphytic colonies of Gloeotrichia) dominated the benthic community. The predominance of nitrogen‐fixing, potentially toxic cyanobacteria likely resulted from excessive nutrient loads and may affect nutrient and trophic dynamics in the river.     

MORPHOLOGICAL AND MOLECULAR CHARACTERIZATION OF PLANKTONIC CYANOBACTERIA FROM BELGIUM AND LUXEMBOURG1

For the first time in Belgium and Luxembourg, the diversity and taxonomy of 95 cyanobacterial strains isolated from freshwater blooms were assessed by the comparison of phenotypes and partial 16S rRNA gene sequences. The results showed the high diversity of nanoplanktonic, picoplanktonic, and benthic–periphytic cyanobacteria accompanying the main bloom‐forming taxa. Indeed, besides 15 morphotypes of bloom‐forming taxa, seven non‐bloom‐forming planktonic morphotypes and 11 morphotypes from benthic–periphytic taxa were isolated in culture from the plankton samples of 35 water bodies. The bloom‐forming strains belonged to the genera Microcystis, Woronichinia, Planktothrix, Anabaena, and Aphanizomenon, whereas the other strains isolated from the same samples were assigned to the nanoplanktonic Aphanocapsa, Aphanothece, Snowella, and Pseudanabaena; to the picoplanktonic Cyanobium; and to the benthic–periphytic Geitlerinema, Komvophoron, Leptolyngbya, Lyngbya, Phormidium, Calothrix, Nostoc, and Trichormus. The results supported both the polyphyletism of genera such as Aphanocapsa, Aphanothece, Leptolyngbya, Geitlerinema, Anabaena, and Aphanizomenon as well as the validity of genera such as Microcystis, Planktothrix, and Pseudanabaena with gas vesicles and cells constricted at the cross wall. The results obtained showed the close relationship between Snowella and Woronichinia for which very few sequences exist. The first sequence of Komvophoron appeared poorly related to other available cyanobacterial sequences. Although in a few cases a good agreement existed between phenotypic and genotypic features, there was generally a discrepancy. Strains with identical morphotypes show small differences in the 16S rRNA sequences, which might be related to the different chemical properties of their habitats. The results showed the importance of the polyphasic approach in order to improve the taxonomy of cyanobacteria.     

GROWTH OF ANTARCTIC CYANOBACTERIA UNDER ULTRAVIOLET RADIATION: UVA COUNTERACTS UVB INHIBITION1

A mat-forming cyanobacterium (Phormidium mur-rayi West and West) isolated from an ice-shelf pond in Antarctica was grown under white light combined with a range of UVA and UVB irradiances. The 4-day growth rate decreased under increasing ultraviolet (UV) radiation, with a ninefold greater response to UVB relative to UVA. In vivo absorbance spectra showed that UVA and to a greater extent UVB caused a decrease in phycocyanin/ chlorophyll a and an increase in carotenoids/chlorophyll a. The phycocyanin/chlorophyll a ratio was closely and positively correlated to the UVB-inhibited growth rate. Under fixed spectral gradients of UV radiation, the growth inhibition effect was dominated by UVB. However, at specific UVB irradiances the inhibition of growth depended on the ratio of UVB to UVA, and growth rates increased linearly with increasing UVA. These results are consistent with the view that UVB inhibition represents the balance between damage and repair processes that are each controlled by separate wavebands. They also underscore the need to consider UV spectral balance in laboratory and field assays of UVB toxicity.     

ULTRASTRUCTURE OF UNICELLULAR N2 FIXING CYANOBACTERIA FROM THE TROPICAL NORTH ATLANTIC AND SUBTROPICAL NORTH PACIFIC OCEANS1

Nitrogen fixing unicellular marine cyanobacteria may have a major role in the global biogeochemistry of N; nevertheless, little is known about their phylogeny and morphology. We isolated N₂ fixing unicellular cyanobacteria from the tropical North Atlantic and subtropical North Pacific Oceans and examined ultrastructural dynamics during dark:light cycles when grown in incubators. The isolate from the subtropical North Pacific was larger and showed a size variation from 3 to 7 μm but had similar morphology and cell division‐plane characteristics as the isolate from the North Atlantic (2.5 μm). Nitrogen fixation only occurred during the dark phase, and ultrastructural analysis demonstrated changes in the appearance and quantity of large carbohydrate‐like granules present in the cells. To verify the composition of these carbohydrate‐like granules, staining with periodic acid, thioacetic acid, and silver was carried out, and a positive reaction was obvious in all cells. The cells from the Atlantic seemed to empty their polysaccharide granules during the night, whereas those from the Pacific showed a decrease in the number of their granules. Our work suggests that phylogenetically related strains of unicellular N₂ fixing cyanobacteria from different oceans showed similar carbohydrate‐like granules that could be used to fuel N₂ fixation during darkness.