CULTURE OF THE TERRESTRIAL CYANOBACTERIUM,NOSTOC FLAGELLIFORME (CYANOPHYCEAE), UNDER AQUATIC CONDITIONS1

Both colonies and free‐living cells of the terrestrial cyanobacterium, Nostoc flagelliforme (Berk. & Curtis) Bornet & Flahault, were cultured under aquatic conditions to develop the techniques for the cultivation and restoration of this endangered resource. The colonial filaments disintegrated with their sheaths ruptured in about 2 days without any desiccating treatments. Periodic desiccation played an important role in preventing the alga from decomposing, with greater delays to sheath rupture with a higher frequency of exposure to air. The bacterial numbers in the culture treated with seven periods of desiccation per day were about 50% less compared with the cultures without the desiccation treatment. When bacteria in the culture were controlled, the colonial filaments did not disintegrate and maintained the integrity of their sheath for about 20 days even without the desiccation treatments, indicating the importance of desiccation for N. flagelliforme to prevent them from being disintegrated by bacteria. On the other hand, when free‐living cells obtained from crushed colonial filaments were cultured in liquid medium, they developed into single filaments with sheaths, within which multiple filaments were formed later on as a colony. Such colonial filaments were developed at 15, 25, and 30° C at either 20 or 60 μmol photons·m^?2·s^?1; colonies did not develop at 180 μmol photons·m^?2·s^?1, though this light level resulted in the most rapid growth of the cells. Conditions of 60 μmol photons·m^?2·s^?1 and 25° C appeared to result in the best colonial development and faster growth of the sheath‐held colonies of N. flagelliforme when cultured indoor under aquatic conditions.     

COMPETITION BETWEEN A PROCHLOROPHYTE AND A CYANOBACTERIUM UNDER VARIOUS PHOSPHORUS REGIMES: COMPARISON WITH THE DROOP MODEL

The ecophysiology and competitive behavior of the prochlorophyte Prochlorothrix hollandica Burger-Wiersma, Stal et Mur, and the cyanobacterium Planktothrix agardhii Anagn. et Kom. were investigated in phosphorus-limited continuous cultures. When the species were exposed to successive saturating pulses of P, the maximal P uptake rate decreased linearly with an increase in the P cell quota. Prochlorothrix had a higher maximal P uptake rate, a lower half-saturation constant for P uptake, higher maximal cell quota for P, and slightly lower minimal cell quota for P than Planktothrix. These data indicate that Prochlorothrix is an affinity and storage strategist, at least when compared to Planktothrix. On the other hand, Prochlorothrix had a lower maximal growth rate than Planktothrix. On the basis of these ecophysiological parameters, we developed a Droop model to predict the time course and outcome of competition under various P regimes. The model predictions were in line with the results of competition experiments under three different P-limited conditions (continuous P supply, 4-day pulse period, 12-day pulse period). Prochlorothrix competitively displaced Planktothrix under both a constant and a pulsed P supply, and competitive displacement of Planktothrix was slowest in the experiment with a 12-day pulse period. In the pulsed experiments, the mean filament lengths and chl a fluorescence of the species oscillated at the same frequency as the pulse additions. In contrast to the Droop model, the predictions of the Monod model were not in line with the outcome of the competition experiments. Our results demonstrate that Prochlorothrix is a very good competitor for P and that the time course and outcome of competition for P in a variable environment can be predicted on the basis of the P uptake and storage characteristics of the species.     

THE FRESHWATER CYANOBACTERIUM PLANKTOTHRIX SP. FP1: MOLECULAR IDENTIFICATION AND DETECTION OF PARALYTIC SHELLFISH POISONING TOXINS

A filamentous cyanobacterium, belonging to the Order of Oscillatoriales, was found to be responsible for a toxic algal bloom in Lake Varese, Italy, during the summer of 1997. Morphological characters, as well as near complete 16S rRNA gene sequencing, revealed that the dominant species of the bloom was most closely related to the genus Planktothrix . In addition, genetic analysis of the phycocyanin operon of Planktothrix sp. FP1 revealed a novel primary structure, previously undescribed within the cyanobacteria, which was used as a genetic marker for rapid detection and identification of this toxic strain. The occurrence of saxitoxin (STX), a principal toxin in paralytic shellfish poisoning (PSP), was confirmed in the natural bloom sample by both pre-column and post-column derivatization high-performance liquid chromatography (HPLC) analyses, and eventually by liquid chromatography/mass spectrometry (LC/MS). The toxicity of this field sample was also revealed by electrophysiological assays in which the extract inhibited 90% of the voltage-dependent Na⁺ current in human neuroblastoma cells at the STX concentration of 80 nM. The cultured strain showed a lower physiologic activity than the bloom sample (67% blockage of Na⁺ current at a toxin concentration of 200 nM), and STX was detected only by pre-column HPLC, indicating the presence of a compound structurally close to STX. Chemical and molecular genetic analyses performed here add Planktothrix sp. FP1 to the growing list of diverse cyanobacterial species capable of synthesizing STX and its related compounds.     

水分胁迫条件下高粱种子的吸水机制及其可溶性糖代谢的变化

以高梁杂交种晋杂８６－１为材料研究了种子吸水萌动过程中胚与胚乳两部分的水分状况及可溶性糖变化的过程。结果表明：在吸水萌动过程中种胚和胚乳在水分及糖代谢方面具有不同的特点。     

武汉东湖磷含量的变动及其分布

本文总结了武汉东湖水体中磷含量（均以PO4计算）的周年逐月季节和年际变动及其分布上的差异（1973－1985年）。按面积加权法计算总磷的平均含量为0．244毫克／升（1983－1985年）,总溶解磷和溶解活性磷的平均含量分别为0．121毫克／升和0．051毫克／升（1981－1984年）,总磷和总溶解磷周年中出现两次高峰含量,即春季（3－5月）和夏末秋初（8－9月）。低含量出现在水温最低的冬季（12－2月）,周年中溶解活性磷高峰含量出现在冬末春初（1－3月）,低含量多数出现在春天夏初（5－7月）。东湖水体中磷含量平面分布有明显的差异,而垂直分布表层和底层差异小,各种形态磷的组成中颗粒磷所占比较最大（1983－1984年平均值）,平均占总磷63．4％,溶解非活性磷所占比较最小,平均占总磷12．0％。     

EFFECT OF ELEVATED TEMPERATURE,DARKNESS, AND HYDROGEN PEROXIDE TREATMENT ON OXIDATIVE STRESS AND CELL DEATH IN THE BLOOM‐FORMING TOXIC CYANOBACTERIUM MICROCYSTIS AERUGINOSA1

This study assessed the implication of oxidative stress in the mortality of cells of Microcystis aeruginosa Kütz. Cultures grown at 25°C were exposed to 32°C, darkness, and hydrogen peroxide (0.5 mM) for 96 h. The cellular abundance, chl a concentration and content, maximum photochemical efficiency of PSII (F_v/F_m ratio), intracellular oxidative stress (determined with dihydrorhodamine 123 [DHR]), cell mortality (revealed by SYTOX‐labeling of DNA), and activation of caspase 3–like proteins were assessed every 24 h. The presence of DNA degradation in cells of M. aeruginosa was also assessed using a terminal deoxynucletidyl transferase‐mediated dUTP nick end labeling (TUNEL) assay at 96 h. Transferring cultures from 25°C to 32°C was generally beneficial to the cells. The cellular abundance and chl a concentration increased, and the mortality remained low (except for a transient burst at 72 h) as did the oxidative stress. In darkness, cells did not divide, and the F_v/F_m continuously decreased with time. The slow increase in intracellular oxidative stress coincided with the activation of caspase 3–like proteins and a 15% and 17% increase in mortality and TUNEL‐positive cells, respectively. Exposure to hydrogen peroxide had the most detrimental effect on cells as growth ceased and the F_v/F_m declined to near zero in less than 24 h. The 2‐fold increase in oxidative stress matched the activation of caspase 3–like proteins and a 40% and 37% increase in mortality and TUNEL‐positive cells, respectively. These results demonstrate the implication of oxidative stress in the stress response and mortality of M. aeruginosa.     

KINETICS OF CELL DEATH IN THE CYANOBACTERIUM ANABAENA FLOS-AQUAE AND THE PRODUCTION OF DISSOLVED ORGANIC CARBON1

Kinetics of cell death and the production of dissolved organic carbon (DOC) were investigated in Anabaena flos-aquae (Lyngb.) Bréb grown on three different N sources (N₂nitrate, and ammonium) in a phosphorus (P)-limited chemostat. The fraction of live cells in the total population increased as growth rate increased with decreasing P limitation. Cell death was less in nitrate and ammonium media than in N₂. The specific death rate (γ), when calculated as the slope ofv^?1_x vs. D^?1, where v_xand D are live cell fraction (or cell viability) and dilution rate, respectively, was 0. 0082 day^?1 in N₂and 0.0042 day^?1 in nitrate. The slope of the plot in ammonium culture was not significant; however, the value of the live cell fraction was within the range for the NO^?₃culture. The fraction of live vegetative cells in N₂ culture was constant at all growth rates and the increase in the overall live cell fraction with growth rate was due entirely to an increase in live heterocysts. Live heterocysts comprised 3.5% of the total cells at a growth rate of 0.25 day^?1 and increased to 6.3% at 0.75 day^?1 with the ratio of live heterocysts to live vegetative cells linearly increasing with growth rate. The fraction of live vegetative cells was invariant in nitrate cultures us in N₂cultures. The live heterocysts fraction also increased with growth rate in nitrate cultures, along with the live heterocysts : live vegetative cells ratio, but the level was lower than in N₂cultures. DOC released from dead cells increased inversely with growth rate in N₂from 36.4% of the total DOC at a growth rate of 0.75 day^?1 to 54.15% at 0.25 day^?1. The contribution of cell death to the total DOC production in nitrate and ammonium media was significantly less than that under N₂DOC from dead cells consisted mainly of high-molecular-weight compounds, whereas DOC excreted from live cells was largely of low molecular weight.     

光强和氮源对念珠藻胞外多糖分泌的影响

胞外多糖（EPS）是结皮蓝藻形成生物结皮的胶结剂,为了理解常球状存在的丝状蓝藻Nostoc胶结沙粒的机理,探讨了光强40、80 E/（m2s）和氮源（气态氮,硝态氮）对结皮优势种Nostoc sp.分泌EPS（包括荚膜多糖CPS和释放多糖RPS）的影响规律及其内在机理。结果发现:Nostoc sp.在气态氮和硝态氮下都有相似的快速生长,但其分泌的RPS、CPS及EPS量,在硝态氮下均随光强的增加而增加,在气态氮下却与光强没有关系。相关代谢研究发现,在硝态氮下细胞内有更高含量的可溶性糖和蔗糖。进一步的相关分析发现,在两种氮源下,蔗糖量与RPS量或CPS量间的显著正相关都只发生在80 E/（m2s）下,在气态氮中,两光强下的胞内总糖量都与CPS量显著负相关。以上结果说明,Nostoc sp.在氮源利用和光强适应方面都有明显优势,它即使在快速生长的对数期,也可同时分泌相当量的EPS,这使其在球状藻殖段形成之前胶结沙粒成为可能。由此可推知,Nostoc sp.在贫瘠沙土表面的最初生长过程中,其胞外的EPS均来自胞内的固碳产物,在高光强下,蔗糖很可能是其EPS合成的原料。       

环境因子对金藻生长和噬藻速率的影响

在微囊藻的大量培养过程中分离到一株能够快速吞噬微囊藻的鞭毛虫-金藻Poterioochromonas sp.,其具有混和营养的特点。研究以人工培养的铜绿微囊藻(Microcystis aeruginosa FACHB469)为饵料,研究了起始饵料浓度、光强、温度和pH等环境因子对Poterioochromonas sp.生长和吞噬饵料速率的影响。结果显示:当无饵料时,金藻的自养生长与光强和温度相关,而与pH无相关性。喂食饵料能显著促进金藻的生长,其吞噬速率和生长速率与起始饵料浓度相关性强,可分别用Michaelis-Menten方程和Monod方程拟合。提供相同量的饵料时,金藻的生长与光强相关性显著,而与温度和pH的相关性不显著;其吞噬速率与pH呈现负相关关系,而与光强和温度相关性不显著。除了在不同pH下的生长外,混合营养时金藻的生长速率与吞噬速率之间存在显著的正相关关系。实验表明适于Poterioochromonas sp.生存并吞噬微囊藻的环境条件较广,这也是进一步探索利用Poterioochromonas sp.控制微囊藻水华的前提。       

叶绿素缺失和异养生长对蓝藻Synechocystis sp.PCC 6803藻胆蛋白含量的影响

通过遮黑培养缺失ｆｒｘＣ基因的蓝藻Ｓｙｎｅｃｈｏｃｙｓｔｉｓｓｐ．ＰＣＣ６８０３突变工程株,获得了叶绿素缺失的藻细胞,吸收光谱测定及数学计算表明,藻细胞中叶绿素缺失后藻胆蛋白含量增加,藻蓝蛋白和别藻蓝蛋白含量分别为相同条件下野生株对照组的４倍和６倍。野生株遮黑培养时,细胞进行异养生长,藻胆蛋白含量下降,藻蓝蛋白和别藻蓝蛋白含量分别为光照培养条件下自养生长的野生株细胞的３４．５％和２５３％。另外,缺失ａｐｃＥ基因的突变工程株细胞的藻胆蛋白含量也少于对照野生株,表明ａｐｃＥ基因的编码蛋白ＬＣＭ与藻胆蛋白的含量相关。     

EFFECTS OF CO2 ENRICHMENT ON THE BLOOM‐FORMING CYANOBACTERIUM MICROCYSTIS AERUGINOSA (CYANOPHYCEAE): PHYSIOLOGICAL RESPONSES AND RELATIONSHIPS WITH THE AVAILABILITY OF DISSOLVED INORGANIC CARBON1

Microcystis aeruginosa Kütz. 7820 was cultured at 350 and 700 μL·L ^{? 1} CO₂ to assess the impacts of doubled atmospheric CO₂ concentration on this bloom‐forming cyanobacterium. Doubling of CO₂ concentration in the airflow enhanced its growth by 52%–77%, with pH values decreased and dissolved inorganic carbon (DIC) increased in the medium. Photosynthetic efficiencies and dark respiratory rates expressed per unit chl a tended to increase with the doubling of CO₂. However, saturating irradiances for photosynthesis and light‐saturated photosynthetic rates normalized to cell number tended to decrease with the increase of DIC in the medium. Doubling of CO₂ concentration in the airflow had less effect on DIC‐saturated photosynthetic rates and apparent photosynthetic affinities for DIC. In the exponential phase, CO₂ and HCO₃ ^? levels in the medium were higher than those required to saturate photosynthesis. Cultures with surface aeration were DIC limited in the stationary phase. The rate of CO₂ dissolution into the liquid increased proportionally when CO₂ in air was raised from 350 to 700 μL·L ^{? 1}, thus increasing the availability of DIC in the medium and enhancing the rate of photosynthesis. Doubled CO₂ could enhance CO₂ dissolution, lower pH values, and influence the ionization fractions of various DIC species even when the photosynthesis was not DIC limited. Consequently, HCO₃ ^? concentrations in cultures were significantly higher than in controls, and the photosynthetic energy cost for the operation of CO₂ concentrating mechanism might decrease.     

EPA高产菌的繁殖体诱导条件初探

研究了诱导介质、处理时间及温度等因素对高产EPA、AA等多不饱和脂肪酸的菌株－－轮梗霉（Diasporangium sp.）产游动孢子能力的影响。结果表明：胡萝卜片介质上、25℃诱导6d,要得到最大数量的孢子。同一条件下游动孢子量则在第8d达到最高。进一步探讨孢子的萌发条件发现：25℃、营养琼脂上孢子的萌发率最高。     

豆乳凝固醇产生菌UV—10发酵条件及其酶学性质的研究

豆乳凝固醇产生菌Bacillus sp.UV-10的最适产酶条件,初始pH6.4,温度26℃,培养时间19h,需要较大的通气量,酶的最适作用pH和温度分别为5．8和70℃,在最适条件下酶活力可达1.84u/mL,pH6.0-7.0稳定性较好,60℃下1h残余酶活60％,Ca^2 ,Fe^2 ,Mg^2 ,Na^2 对其有较强的激活作用,而Zn^2 ,Al^3 则有抑制作用。     

ELECTRON MICROSCOPE OBSERVATIONS ON THE INTERACTION OF BDELLOVIBRIO BACTERIOVORUS WITH PHORMIDIUM LURIDUM AND SYNECHOCOCCUS SP. (CYANOPHYCEAE)1

The effects of Bdellovibrio bacteriovorus (Stolp & Starr) culture supernatants on Phormidium luridum var. olivacea Boresch and Synechococcus sp. were examined by transmission electron microscopy. Both normal (nonheat-treated) and heat-treated bdellovibrio supernatant caused the formation of intrathylakoidal vesicles in P. luridum in 24–48 h. This vesiculation increased until 96–129 h when the P. luridum showed loss of the mucopeptide layer in the cell envelope and subsequently lysed. Similar treatment of Synechococcus sp. with the bdellovibrio supernatants showed a different ultrastructural pattern with the apparent dissolution of many of the photosynthetic membranes in the bluegreen cells. Myelin-like membranous configurations were seen in some of these treated cells. The results suggest that an autolytic mechanism in P. luridum and Synechococcus sp. is stimulated by the bdellovibrio secretions.     

有机磷农药对铜绿微囊藻生长及摄磷效应的动力学研究

研究了两种有机磷农药 (甲胺磷、辛硫磷 )在较低浓度时对铜绿微囊藻的生长效应以及微囊藻摄取磷形态 :总溶解磷 (TSP)、溶解反应磷 (SRP)和溶解有机磷 (DOP)的动力学规律。保持各培养基中总磷的浓度 6 95mg/L ,与培养基中不加有机磷农药相比 ,当培养基中甲胺磷与K2 HPO4的浓度比为 1∶11和 1∶9,辛硫磷与K2 HPO4的浓度比为1∶349和 1∶114时 ,微囊藻的生长受到促进 ,并且当甲胺磷与K2 HPO4,的浓度比为 1∶9,辛硫磷与K2 HPO4的浓度比为1∶114时促进效果最好 ,而当培养基中没有K2 HPO4甲胺磷作为惟一磷源时 ,微囊藻的生长受到明显的抑制。微囊藻摄取SRP的速度快于其他磷形态 ,而对DOP的利用较少。微囊藻生长越快 ,对SRP的摄取速率也越快。结果表明低浓度有机磷农药促进微囊藻生长的主要原因是有机磷的加入促进了藻类对SRP的摄取     

EFFECTS OF IRON AND LIGHT STRESS ON THE BIOCHEMICAL COMPOSITION OF ANTARCTIC PHAEOCYSTIS SP. (PRYMNESIOPHYCEAE). II. PIGMENT COMPOSITION

A strain of Phaeocystis sp., isolated in the Southern Ocean, was cultured under iron- and light-limited conditions. The cellular content of chlorophyll a and accessory light-harvesting (LH) pigments increased under low light intensities. Iron limitation resulted in a decrease of all light-harvesting pigments. However, this decrease was greatly compensated for by a decrease in cell volume. Cellular concentrations of the LH pigments were similar for both iron-replete and iron-deplete cells. Concentrations of chlorophyll a were affected only under low light conditions, wherein concentrations were suppressed by iron limitation. Ratios of the LH pigments to chlorophyll a were highest for iron-deplete cells under both light conditions. The photoprotective cycle of diato/diadinoxanthin was activated under high light conditions, and enhanced by iron stress. The ratio of diatoxanthin to diadinoxanthin was highest under high light, low iron conditions.   Iron limitation induced synthesis of 19'-hexanoyloxyfucoxanthin and 19'-butanoyloxyfucoxanthin at the cost of fucoxanthin. Fucoxanthin formed the main carotenoid in iron-replete Phaeocystis cells, whereas for iron-deplete cells 19'-hexanoyloxyfucoxanthin was found to be the main carotenoid. This shift in carotenoid composition is of importance in view of the marker function of both pigments, especially in areas where Phaeocystis sp. and diatoms occur simultaneously. A hypothesis is presented to explain the transformation of fucoxanthin into 19'-hexanoyloxyfucoxanthin and 19'-butanoyloxyfucoxanthin, referring to their roles as a light-harvesting pigment.     

GROWTH AND NITROGEN FIXATION OF THE DIAZOTROPHIC FILAMENTOUS NONHETEROCYSTOUS CYANOBACTERIUM TRICHODESMIUM SP. IMS 101 IN DEFINED MEDIA: EVIDENCE FOR A CIRCADIAN RHYTHM1

Trichodesmium sp. IMS 101, originally isolated from coastal western Atlantic waters by Prufert-Bebout and colleagues and maintained in seawater-based media, was successfully cultivated in two artificial media. Its characteristics of growth, nitrogen fixation, and regulation of nitrogen fixation were compared to those of natural populations and Trichodesmium sp. NIBB 1067. Results indicate that the culture grown in artificial media had nitrogen fixation characteristics similar to those when the culture is grown in seawater-based medium and to those of Trichodesmium sp. in the natural habitat. The study provides practical artificial media to facilitate the physiological studies of these important diazotrophic cyanobacteria, as well as the cultivation of other Trichodesmium species in future studies. Manipulations of the light/dark cycle were performed to determine whether or not the daily cycle of nitrogen fixation is a circadian rhythm. Cultures grown under continuous light maintained the cycle for up to 6 days. We demonstrated that the daily cycle of nitrogen fixation in Trichodesmium sp. IMS 101 was at least partially under the control of a circardian rhythm.     

ISOLATION OF ccmKLMN GENES FROM THE MARINE CYANOBACTERIUM, SYNECHOCOCCUS SP. PCC7002 (CYANOPHYCEAE), AND EVIDENCE THAT CcmM IS ESSENTIAL FOR CARBOXYSOME ASSEMBLY

A high CO₂ requiring mutant of the marine cyanobacterium Synechococcus PCC7002 was generated using a random gene-tagging procedure. This mutant demonstrated a reduced photosynthetic affinity for inorganic carbon (C_i) and accumulated high internal levels of C_i that could not be used for photosynthesis. Analysis of the mutant genomic DNA showed that the mutagenesis had disrupted a cluster of genes involved in the cyanobacterial CO₂ concentrating mechanism (CCM), the so-called ccm genes. These characteristics are consistent with a cyanobacterial mutant with defects in carboxysome assembly and/or functioning. Further genomic analyses indicated that the genes of the Synechococcus PCC7002 operon, ccmKLMN , are structurally similar to those of two closely related cyanobacteria, Synechococcus PCC7942 and Synechocystis PCC6803. The Synechococcus PCC7002 ccmM gene, which encodes a polypeptide with a predicted size of 70 kDa, was the direct target of the mutagenesis event. The CcmM protein has two distinct regions: an N-terminal region that shows similarity to an archaeon gamma carbonic anhydrase and a C-terminal region that contains repeated domains demonstrating sequence similarity to the small subunit of Rubisco. Physiological analysis of a ccmM -defined mutant showed that these cells were essentially identical to the original mutant; they required high CO₂ concentrations for growth, they had a low photosynthetic affinity for C_i, and they internalized C_i to high levels. Moreover, ultrastructural examination showed that both the original and the defined mutants lack carboxysomes. Thus, our results demonstrate that the ccmM gene of Synechococcus PCC7002 encodes a polypeptide that is essential for carboxysome assembly and therefore for proper functioning of the cyanobacterial CCM.